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Cytotoxicity of Silver Nanoparticles Synthesized by Cytotoxicity of Silver Nanoparticles Synthesized by

Cytotoxicity of Silver Nanoparticles Synthesized by - PowerPoint Presentation

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Cytotoxicity of Silver Nanoparticles Synthesized by - PPT Presentation

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

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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

Slide2

Research 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

Slide3

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

Slide4

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.= Downregulated genes= Upregulated genes

= S-phase arrest-induced genes

Gene expression profiling of

C.

roseus

-treated

Jurkat

cells

Slide5

Introduction

Slide6

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 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)

Slide7

Problem 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

Slide8

MethodologyMTS

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

Slide9

Results 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

Slide10

Proliferative 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

Slide11

Proliferative 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

Slide12

Proliferative 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

Slide13

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

 anti-tubulin properties  inhibit formation of mitotic spindles by damaging microtubules  cell cycle arrest

[23]

Slide14

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, α-tocopherol, reduced glutathione and antioxidation enzymes)  scavenge the ROS [25]

Slide15

Expression 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.

Slide16

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.

Slide17

Expression 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. **

Slide18

Expression 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.

Slide19

At 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

Slide20

Effects 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.

Slide21

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.

Slide22

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 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

Slide23

Jurkat 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

Slide24

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Slide26

Acknowledgement

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

Slide27

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