SEMINAR ON

TARGETING INTRINSIC PATHWAY OF

APOPTOSIS BY PIPERLONGUMINE, A
NATURALCOMPOUND FOR ANTICANCER
THERAPY

Under the supervision ofDr. Sistla Ramakrishna

Principal Scientist ,
Pharmacology Division,
IICT, Hyderabad.

Presented by
A.Hasitha Shilpa
PT/2010/07
M.S. Pharm (IV sem)

CONTENTS:

INTRODUCTION
LITERATURE REVIEW
OBJECTIVE OF WORK
WORK DONE AND RESULTS
CONCLUSION
REFERENCES

5/8/15

INTRODUCTION:
CANCER-APOPTOSIS-ROS

APOPTOSIS : DEAD MANS

HANDLE

Apoptosis is a form of cell death in which an individual cell

undergoes an internally controlled or programmed
transition from an intact metabolically active state into a
number of shrunken remnants that retain their membrane
bound integrity.

Apoptotic cell death (type I PCD) (from Greek Apo,

meaning from, and ptosis', meaning falling) is presented
by :
Shrinkage of cell,
Membrane blebbing and
Phagocytes fall from the dying cell into apoptotic bodies
Cancer Letters 300 (2011) 105114
4

5/8/15

CANCER-APOPTOSIS:
EXTRINSI
C

INTRINSI
C

Cancer Letters 300 (2011) 105

114

5/8/15

APOPTOSIS-ROS:

Apoptosis 2000; 5: 415418

5/8/15

LITERATURE REVIEW

5/8/15

REPORTED WORK ON
PIPERLONGUMINE:

Selective killing of cancer cells by a small molecule

targeting the stress response to ROS.
(Lakshmi Raj et.al., 2011)
Runaway ROS as a Selective Anticancer Strategy.
(Elizabeth I. Parkinson and Paul
Hergenrother,2011)

J.

Overview for Various Aspects of the Health Benefits

of Piper Longum Linn. Fruit.
(Suresh Kumar, Jitpal Kamboj, Suman, Sunil
Sharma; 2011)

5/8/15

PIPERLONGUMI
NE :

Piperlongumine reduced the viability of 14 cancer cell

lines of different origins, with an average IC50 value
of ~ 7 M, but did not affect six different
noncancerous cell types.

The two highest hits were glutathione S-transferase pi 1

(GSTP1) and carbonyl reductase (CBR1), both of which are
known to detoxify xenobiotics.
ChemMed Chem 2011, 6, 1957 1959
9

5/8/15

OBJECTIVE OF THE
WORK:

To target extrinsic and intrinsic pathway of apoptosis

by natural products for anticancer therapy

To
evaluate
the
antiproliferative
activity
of
Piperlongumine, and its effect on cell growth and
apoptosis on human lung cancer cell line (A549).

To examine the role of ROS (Reactive Oxygen

Species), Mitochondria, Bcl-2 family proteins (Bax,
Bcl-2),
&
caspases
in
the
regulation
of
Piperlongumine mediated apoptosis

10

5/8/15

WORK DONE
AND
RESULTS

11

5/8/15

CYTOTOXICITY ASSAYS

MTT ASSAY :

CELL LINE

IC50 values of Piperlongumine on different cancer cell lines.

TEST
COMPOUND

ORIGIN

DOXORUBICIN

IC50(M)

A549

LUNG CANCER

3.98

<1

ACHN

RENAL CANCER

2.42

<1

MCF-7

BREAST CANCER

2.75

<1

COLO205

COLON CANCER

9.0

< 0.1

HT-29

COLON CANCER

5.25

< 0.1

HEK

NON-CANCEROUS

13

IC50(M)

5/8/15

LDH
Assay :

Piperlongumine treated cells showed increase in LDH

activity due to lysis of the cell and release of LDH
indicating Apoptosis.
14

5/8/15

CALCEIN AM ASSAY:

CALCEIN AM STD GRAPH

2000
1800
1600
1400

f(x) = 0.07x + 512.52

R = 0.91

1200
1000
Absorbance
800
600
400
200
0
0

5000

10000 15000 20000 25000

No.of cells

Piperlongumine treated cells showed gradual decrease

in the viable cells in comparison to control cells
15

5/8/15

Bioluminescent Assay for ADP/ATP Ratio:

CONCENTRATION

ADP/ATP RATIO

CONTROL

0.099835

2 M

0.242626

4 M

0.715311

Increased levels of ATP and decreased levels of ADP

signify proliferating cells. Conversely, decreased
levels of ATP and increased levels of ADP represent
16
apoptotic cells.

5/8/15

Effect of Piperlongumine on Morphological changes in A549 cells :

CONTROL

TREATED

Cell shrinkage, loss of morphology and

contact between the cells was observed
in the treated cells. 17

5/8/15

NUCLEAR CONDENSATION PROPERTY OF PIPERLONGUMINE :

(A)ACRIDINE ORANGE/ETHIDIUM BROMIDE STAINING :

CONTROL
TREATED

Green cells with intact membrane indicates live cells,

Orange cells : Acridine orange indicates early apoptotic
cells, Ethidium bromide indicates late apoptotic cells.
18

5/8/15

(B) PROPIDIUM IODIDE STAINING :

CONTROL
TREATED

Control cells don't show any fluorescence whereas

treated shows red fluorescence due to Nuclear
19
condensation.

5/8/15

DNA FRAGMENTATION:
L
4M

2M

Piperlongumine
showed
DNA
fragmentation
in
A549
cells
when
compared to control
cells.

L Ladder; C- Control;
20

5/8/15

FLOW -CYTOMETRIC CELL CYCLE ANALYSIS:

The mechanism of cell cycle arrest of the test

compound(piperlongumine) was analysed using
FLOURESCENCE ACTIVATED CELL SORTER(FACS)
ANALYSIS.
Propidium iodide(PI) is a fluorogenic compound
binds to nucleic acids and is proportional to DNA
content of a cell.
Apoptotic cells displayed a broad hypo diploid
peak(sub-G1 peak) differentiated from cells with
normal(diploid) DNA content .

21

5/8/15

CONTROL

1M

2M

FLOW CYTOMETRY GRAPHS:

4
M

STD

22

Dose-dependent
increase in Sub-G1
peak
(R5)
was
observed.

5/8/15

GRAPHICAL REPRESENTATION OF FLOWCYTOMETRY

DATA:

Piperlongumine showed increased DNA content and resulted

23 increase in concentration.
5/8/15
in increased Sub G1 Peak with

Annexin V-FITC Assay

:
Annexin V-FITC is detected as a Green fluorescence and
Propidium iodide is detected as a Red fluorescence.
The annexin V assay has been
widely accepted as a marker of
apoptosis

Annexin V is a Ca++dependent
phospholipidbinding protein that binds
strongly to phosphatidylserine
residues on the cell membrane.

24

5/8/15

ANNEXIN V FITC/PROPIDIUM IODIDE STAINING:

CONTROL

TREATED-2M

TREATED4M

Outer green membrane of the cells indicate Early

apoptosis(Annexin V stain)
Outer green with inner red fluorescence indicates Late
25 and Propidium iodide staining)
5/8/15
apoptotic cells (Annexin V FITC

ANNEXIN V FITC ANALYSIS:

FLOWCYTOMETRY
GRAPHS:

CONTROL

Piperlongumine
treated
cells
showed dose-dependent significant
percentage increase in Early and
Late apoptosis.

1M

2M

26

4M

5/8/15

MEASUREMENT OF ROS PRODUCTION:

The intracellular ROS can be estimated by fluorescent

dye 2,7- Dichlorofluorescein diacetate (DCFH-DA).

Thus, the DCF fluorescence intensity is directly

proportional to the amount of ROS produced
27
intracellularly .

5/8/15

PRODUCTION OF REACTIVE OXYGEN

SPECIES:

CONTROL

TREATED
4M

Green fluorescence indicates the production of ROS in

treated cells whereas control
do not show any green
28
fluorescence.

5/8/15

MEASUREMENT OF REACTIVE OXYGEN SPECIES:

Emission Scan

Very significant increase

in ROS production in
Piperlongumine
treated
cells.

29

5/8/15

EVALUATION OF JC-1 STAINED CELLS BY FLUORESCENCE

MICROSCOPY:

CONTROL

TREATED(4M)

Green fluorescence : decrease in m in treated cells

Red fluorescence : higher m30

5/8/15

DETERMINATION OF m BY BIOTEK PLATE READER:

Significant decrease in Red/Green ratio indicates

lowering of m .
31

5/8/15

DETERMINATION OF INTRACELLULAR Ca2+ LEVELS USING FLUO3AM:

Increased calcium levels with increase in concentrations

directly proportional to the level of apoptosis and ROS
32
generation by Piperlongumine.

5/8/15

DNA QUANTIFICATION USING HOECHST 33258:

CONTROL

TREATED(4M)

The morphology of apoptotic cells is readily apparent, as

shown by cell shrinkage and condensed chromatin was
33
5/8/15
observed in the Piperlongumine
treated cells in comparison
to Control cells.

MEASUREMENT OF DNA QUANTIFICATION BY SPECTROFLUORIMETRY:

DNA QUANTIFICATION USING HOECHST

23000
22500
22000
21500
21000
20500
RFU
20000
19500
19000
18500
18000
CTL

Concentration(M)

Piperlongumine resulted in dose dependent DNA

quantification levels in comparison to control cells.
34

5/8/15

ESTIMATION OF GSH LEVELS :

Piperlongumine treated higher concentrations

showed significant decrease in GSH levels
35
determined using ONEWAY-ANOVA.

5/8/15

ESTIMATION OF CATALASES ACTIVITY

:

Piperlongumine treated cells showed dose-dependent

increased %H2O2(V/V) that indicates decreased
36
5/8/15
catalases activity .

ESTIMATION OF SUPEROXIDE DISMUTASE ACTIVITY:

Piperlongumine treated cells showed dose dependent decrease

in %O22-Inhibition comparison to control cells. Pre-treatment with
2Nac
showed
increased
%O
activity.(Standard
37 2 Inhibitory
5/8/15
reference)

MEASUREMENT OF SUPEROXIDE ANION PRODUCTION:

Piperlongumine treated cells showed dose dependent

increase in superoxide anion production in comparison
38
5/8/15
to control cells.

MATRIX METALLOPROTEINASE-2 INHIBITION:

Piperlongumine showed dose dependent increase in

MMP2 inhibition with respect to the relative
fluorescence units in39comparison to control cells. 5/8/15

MATRIX METALLOPROTEINASE-9 INHIBITION:

Piperlongumine showed dose dependent increase in

MMP9 inhibition with respect to the relative
fluorescence units in comparison to control cells.
40

5/8/15

ESTIMATION OF CASPASE-3 ACTIVITY:

Piperlongumine showed dose-dependent increase in

Caspase-3 expression and was confirmed by addition of
inhibitor.
41

5/8/15

CONCLUSION:

In this study, we analyzed apoptosis and antiproliferative

effects
of
Piperlongumine elucidate this probable
mechanism of action in lung cancer cells.(A549)
The proposed mechanism of Apoptosis is as follows :
Apoptosis is associated with the generation of ROS,
depletion of GSH, increase in intracellular calcium level,
increase in ADP/ATP ratio, loss of mitochondrial membrane
potential, imbalance between pro-apoptotic & antiapoptotic proteins and activation of caspase-3.
Further experimentation on Piperlongumine in animal
models is essential to take this compound further for
clinical use.

42

5/8/15

PROPOSED MECHANISM OF PIPERLONGUMINE:

43

5/8/15

REFERENCES:

Alan L. Harvey;Natural products in drug discovery.Drug Discovery Today, 2008,13,19/20.

A.Chaiteriee and C. P. Durr; alkaloids of Piper longum linn-i structure and synthesis of piperlongumine
And piperlonguminine.Tetrahedron,1967,23,1769-1781.
Allen TD: Ultrastructural aspects of cell death. In: Perspectives on Mammalian Cell Death, Potten CS (eds). Oxford
University Press,Oxford, 1987, 59,3965.
BOEHRINGER MANNHEIM;Apoptosis and Cell Proliferation 2ndedition.
Baran et al.,Baran, C.P., Zeigler, M.M., Tridandapani, S., Marsh, C.B. The role of ROS and RNS in regulating life and
death of blood monocytes. Curr. Pharm. Des., 2004,10, 855866.
Christian Bailly;Ready for a comeback of natural products in oncology.Biochemical pharmacology,2009, 77 ,1447
1457.
Chatterjee A, Dutta CP. Alkaloids of Piper longumLinn. I. Structure and synthesis of piperlongumine and piperlonguminine.Tetrahedron.1967;23:176981.
D N Dhanasekaran and E P Reddy; JNK signaling in apoptosis JNK and apoptosis.Oncogene, 2008,27, 6245-6251 .
Demetrius Matassov, Terri Kagan, Julie Leblanc, Marianna Sikorska, and Zahra Zakeri; Measurement of Apoptosis by
44
5/8/15
DNA Fragmentation. Methods in Molecular Biology, 282,1-17.

Eric Miller;Apoptosis Measurement by Annexin V Staining.Methods in Molecular Medicine,191-202.

Gary A. Piazza, Alanna L. Kulchak Rahm, Mary Krutzsch, et al.Antineoplastic Drugs Sulindac Sulfide and Sulfone
Inhibit CellGrowth by Inducing Apoptosis. Cancer Res,1995;55:3110-3116.
Guohong Zhang, V anessaGurtu, Steven R. Kain and Guochen Yan;Early Detection of Apoptosis Using a
Fluorescent Conjugate of Annexin V. BioTechniques ,1997,23:525-531.
JamilZargan, Sadiq Umar, Mir Sajad, M. Naime, Shakir Ali, Haider A. Khan;Scorpion
venom(Odontobuthusdoriae) induces apoptosis by depolarization of mitochondria and reduces S-phase population
in humanbreast cancer cells (MCF-7).Toxicology in Vitro,2011,25,17481756.
Jane L. Watson et.al., Curcumin causes superoxide anion production and p53 independent apoptosis in human
colon cancercells.2010.
KAMIYA BIOMEDICAL COMPANY; SOD Assay.
Lakshmi Raj et.al., Selective killing of cancer cells by a small molecule targeting the stress response to ROS.
Research letter,Nature,2011,475,231-234.
Lee et.al., United States Patent Publication.2009.
45

5/8/15

Magdalena L. Circuand Tak Yee Aw; Reactive oxygen species, cellular redox systems and apoptosis. Free Radic Biol
Med. 2010 ,48(6): 749762.
Mohd Fadzelly Abu Bakar. Maryati Mohamad, Asmah Rahmat, Steven A. Burr, Jeffrey R. Fry.. Cytotoxicity, cell cycle
arrest, and apoptosis in breast cancer cell lines exposed to an extract of the seed kernel of Mangifera pajang
(bambangan). Food and Chemical Toxicology. 2010,48.16881697.
MMP-2 Fluorimetric Drug Discovery Kit BML-AK409;Enzo Life Sciences.
MMP-9 Fluorimetric Drug Discovery Kit BML-AK411; Enzo Life Sciences.
Nicholson, D.W., et al., Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis.
Nature, 1995,376, 37-43 .
Richard M. Locksley,Nigel Killeen, and Michael J. Lenardo; The TNF and TNF Receptor Review Superfamilies:
Integrating Mammalian Biology. Cell, 2001, 104, 487501.
Susan Elmore;Apoptosis: A Review of Programmed Cell Death. Toxicol Pathol. 2007 ; 35, 495516.

46

5/8/15

VerenaJendrossek; Targeting apoptosis pathways by Celecoxib in cancer. Cancer Letters ,2011.

Wang, X.M.et al. ;H. Immunol. 1993,37(4):264.
Van Engeland,M.,Nieland,I.,J.W.,Ramackers,F.C.S.,et.al. Annexin V-affinity assay: A review on an apoptosis detection
system based on phosphotidylserine exposure. Cytometry,1998,31,1-9.
Zorov, D.B., Juhaszova, M., Sollott, S.J.,. Mitochondrial ROS-induced ROSrelease: an update and review.
Biochim.Biophys.Acta,2006,1757, 509517.

47

5/8/15

ACKNOWLEDGEMENT
S:
Dr. Ahmed Kamal
Dr. Sistla Ramakrishna
Mr. T.Venu
Dr.V.G.M.Naidu
Mr. Kulkarni
NIPER Faculty
IICT Labmates
NIPER Friends
48

5/8/15

Thank
you

50

5/8/15

51

5/8/15

52

5/8/15