PowerPoint Presentation

TARGETING INTRINSIC PATHWAY OF APOPTOSIS BY PIPERLONGUMINE, A NATURALCOMPOUND FOR ANTICANCER THERAPY

SEMINAR ONUnder the supervision of-

Dr. Sistla Ramakrishna Principal Scientist , Pharmacology Division,IICT, Hyderabad.

Presented byA.Hasitha ShilpaPT/2010/07M.S. Pharm (IV sem)12-Jun-12INTRODUCTION

LITERATURE REVIEW

OBJECTIVE OF WORK

WORK DONE AND RESULTS

CONCLUSION

REFERENCESCONTENTS:6/12/20122CANCER-APOPTOSIS-ROSINTRODUCTION: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 bodiesCancer Letters 300 (2011) 105114

6/12/2012412-Jun-12CANCER-APOPTOSIS:

EXTRINSICINTRINSICCancer Letters 300 (2011) 1051146/12/2012512-Jun-12APOPTOSIS-ROS:

6/12/20126Apoptosis 2000; 5: 415418LITERATURE REVIEW6/12/20127REPORTED 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 J. Hergenrother,2011)

Overview for Various Aspects of the Health Benefits of Piper Longum Linn. Fruit. (Suresh Kumar, Jitpal Kamboj, Suman, Sunil Sharma; 2011)

6/12/20128PIPERLONGUMINE :

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

6/12/20129To 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 apoptosisOBJECTIVE OF THE WORK:

6/12/201210WORK DONE AND RESULTS6/12/201211CYTOTOXICITY ASSAYSCELL LINEORIGIN TEST COMPOUND IC50(M) DOXORUBICINIC50(M)A549LUNG CANCER3.98< 1ACHNRENAL CANCER2.42< 1MCF-7BREAST CANCER2.75< 1COLO205COLON CANCER9.0< 0.1HT-29COLON CANCER5.25< 0.1HEKNON-CANCEROUS --MTT ASSAY : IC50 values of Piperlongumine on different cancer cell lines.6/12/201213MTT assay is a colorimetric assay that measures the reduction of MTT by mitochondrial succinate dehydrogenase. MTT reagent enters the cells and passes into the mitochondria where it is reduced to an insoluble, coloured formazan product. The formazan product is then solubilised with an organic solvent (DMSO or isopropanol) and the intensity of colour is measured by spectrophotometer. The amount of colour produced is directly proportional to the number of viable cells

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LDH Assay :Piperlongumine treated cells showed increase in LDH activity due to lysis of the cell and release of LDH indicating Apoptosis.

6/12/201214LDH was determined by Caymans Lactate dehydrogenase is a soluble enzyme located in cytosol. The enzyme is released into the surrounding culture medium upon cell damage or lysis, processes that occur during apoptosis. Hence LDH activity in cell culture medium can be Indicator of cell membrane integrity and thus the measurement of cytotoxicity. Since the activity of intracellular LDH corresponds to the number of cells in the culture, quantification of LDH in cell lysate can be used as a measurement of cell involves.This assay involves two steps :In first step LDH catalyses the reduction of NAD+ to NADH and H+ by oxidation of lactate to pyruvate.In second step reaction diaphorase uses the newly formed NADH and H+ to catalyse the reduction of a tetrazolium salt(INT) to highly coloured formazan.(490nm absorbance)12-Jun-126/12/201215

CALCEIN AM ASSAY:Piperlongumine treated cells showed gradual decrease in the viable cells in comparison to control cells

CONCENTRATIONADP/ATP RATIO CONTROL 0.099835 2 M 0.242626 4 M 0.715311 Bioluminescent Assay for ADP/ATP Ratio:Increased levels of ATP and decreased levels of ADP signify proliferating cells. Conversely, decreased levels of ATP and increased levels of ADP represent apoptotic cells.

6/12/201216Increased levels of ATP and decreased levels of ADP signify proliferating cells. Conversely, decreased levels of ATP and increased levels of ADP represent apoptotic or necrotic cells where the decrease in ATP and increase in ADP is much more pronounced in necrosis versus apoptosis. ADP/ATP Ratio =RLU C RLU B/RLU A ; ATP + D-luciferin + O2 gives (Luciferase)oxyluciferin + AMP + PPi+ CO2+ light

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Effect of Piperlongumine on Morphological changes in A549 cells :

CONTROL TREATEDCell shrinkage, loss of morphology and contact between the cells was observed in the treated cells.

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NUCLEAR CONDENSATION PROPERTY OF PIPERLONGUMINE :

ACRIDINE ORANGE/ETHIDIUM BROMIDE STAINING :

CONTROL TREATEDGreen cells with intact membrane indicates live cells, Orange cells : Acridine orange indicates early apoptotic cells, Ethidium bromide indicates late apoptotic cells.

6/12/201218Acridine orange is taken up by both viable and dead cells. It would fluoresce green when bound to double stranded DNA in live cells and fluoresce orange when bound to single stranded DNA which dominates in dead cells. Ethidium bromide was excluded from living cells. However, late apoptotic or necrotic cells have ruptured membrane that allows the entrance of ethidium bromide to intercalate into DNA and fluoresce orange.12-Jun-12

(B) PROPIDIUM IODIDE STAINING :

CONTROL TREATED Control cells don't show any fluorescence whereas treated shows red fluorescence due to Nuclear condensation.

6/12/201219Propidium Iodide is the most commonly used dye to quantitatively assess DNA content. PI is membrane impermeant and generally excluded from viable cells. PI is commonly used for identifying dead cells.12-Jun-12

L C 2M 4M L Ladder; C- Control; DNA FRAGMENTATION:Piperlongumine showed DNA fragmentation in A549 cells when compared to control cells.

6/12/201220DNA fragmentation has been shown to result from activation of an endogenous Ca2+ and Mg2+ dependent nuclear endonuclease. This enzyme selectively cleaves DNA at sites located between nucleosomal units (linker DNA) generating mono- and oligonucleosomal DNA fragments by piperlongumine.12-Jun-12The 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 .

FLOW -CYTOMETRIC CELL CYCLE ANALYSIS:

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CONTROL1M2M4MSTDFLOW CYTOMETRY GRAPHS:Dose-dependent increase in Sub-G1 peak (R5) was observed.6/12/201222Piperlongumine showed increased DNA content and resulted in increased Sub G1 Peak with increase in concentration.

GRAPHICAL REPRESENTATION OF FLOWCYTOMETRY DATA:

6/12/201223A reduced cellular DNA content due to fractioning of the apoptotic cells & formation of apoptotic bodies is a characteristic feature of cells undergoing apoptosis. This leads to occurrence of sub-G1 cell population with higher DNA content compared to cells in the G1 phase of the cell cycle.12-Jun-12Annexin 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 phospholipid-binding protein that binds strongly to phosphatidylserine residues on the cell membrane.

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ANNEXIN V FITC/PROPIDIUM IODIDE STAINING:CONTROLTREATED-2MTREATED-4M Outer green membrane of the cells indicate Early apoptosis(Annexin V stain) Outer green with inner red fluorescence indicates Late apoptotic cells (Annexin V FITC and Propidium iodide staining)

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FLOWCYTOMETRY GRAPHS:ANNEXIN V FITC ANALYSIS:CONTROL1M2M4MPiperlongumine treated cells showed dose-dependent significant percentage increase in Early and Late apoptosis.

6/12/201226The 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 intracellularly .MEASUREMENT OF ROS PRODUCTION:

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CONTROLTREATED 4MPRODUCTION OF REACTIVE OXYGEN SPECIES: Green fluorescence indicates the production of ROS in treated cells whereas control do not show any green fluorescence.

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MEASUREMENT OF REACTIVE OXYGEN SPECIES:Very significant increase in ROS production in Piperlongumine treated cells.

Emission Scan6/12/201229The intracellular ROS can be estimated by fluorescent dye 2,7- Dichlorofluorescein diacetate (DCFH-DA). This dye is a stable nonpolar compound that readily diffuses into cells and is hydrolyzed by intracellular esterase to yield non-fluorescent Dichlorofluorescein (DCFH), which is trapped within the cells & is oxidized to fluorescent DCF in presence of ROS. Thus, the DCF fluorescence intensity is directly proportional to the amount of ROS produced intracellularly. Ex485 nm and Em530 nm.

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EVALUATION OF JC-1 STAINED CELLS BY FLUORESCENCE MICROSCOPY:CONTROLTREATED(4M)

Green fluorescence : decrease in m in treated cellsRed fluorescence : higher m

6/12/201230In healthy cells with high mitochondrial m, JC-1 spontaneously forms complexes known as J-aggregates with intense red fluorescence. On the other hand, in apoptotic or unhealthy cells with low m, JC-1 remains in the monomeric form, which shows only green fluorescence. The ratio of green to red fluorescence is dependent only on the membrane potential .12-Jun-12DETERMINATION OF m BY BIOTEK PLATE READER: Significant decrease in Red/Green ratio indicates lowering of m .

6/12/201231The mitochondrial Trans membrane potential is expressed as JC-1 fluorescence units in terms of red fluorescence to green fluorescence ratio. The cells without drug treatment showed increased red/green fluorescence signal ratio, which corresponds to the polarised mitochondria. Drug incubation of the cells for 1h significantly reduced the ratio. This decreased ratio in red/green signal after drug treatment signifies an alteration in mitochondrial membrane potential. Control cells displayed a high Red/Green fluorescence, where as treated cells displayed a low Red/Green fluorescence as JC-1 dye was converted to a green fluorescent monomeric form due to fall in mitochondrial membrane potential indicating apoptosis.Red fluorescence at Ex550 nm and Em590 nm and for green fluorescence at Ex490 nm and Em535 nm.

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DETERMINATION OF INTRACELLULAR Ca2+ LEVELS USING FLUO3AM: Increased calcium levels with increase in concentrations directly proportional to the level of apoptosis and ROS generation by Piperlongumine.

6/12/201232When cells become irreparably damaged or otherwise need to undergo apoptosis, the mitochondria release small amounts of a substance known as cytochrome c. The cytochrome c causes the endoplasmic reticulum to release its calcium, resulting in an increase in intracellular calcium. This increase in calcium triggers other chemical changes, including the release of more cytochrome c, which induce apoptosis .ROS also evokes increased [Ca2+] in the absence of extracellular calcium, indicating that ROS mobilizes calcium from intracellular stores leading cells into an apoptotic state Intracellular calcium was determined by Fluorescent probe Fluo-3AM (Invitrogen). Fluo-3AM enters cell as a membrane permeable acetoxymethyl (AM) ester. Once inside the cell, Fluo-3AM is hydrolyzed by intracellular esterases. Cell membrane impermeable, negatively charged form of Fluo-3AM is now capable of binding with Ca2+.[490-520nm]12-Jun-12

DNA QUANTIFICATION USING HOECHST 33258:CONTROLTREATED(4M) The morphology of apoptotic cells is readily apparent, as shown by cell shrinkage and condensed chromatin was observed in the Piperlongumine treated cells in comparison to Control cells.

6/12/201233The dye, weakly fluorescent itself in solution, binds specifically to the A-T base pairs in dsDNA resulting in an increase in fluorescence and a shift in the emission maximum from 500 to 460 nm. 12-Jun-12MEASUREMENT OF DNA QUANTIFICATION BY SPECTROFLUORIMETRY:Piperlongumine resulted in dose dependent DNA quantification levels in comparison to control cells.

6/12/201234The dye, weakly fluorescent itself in solution, binds specifically to the A-T base pairs in dsDNA resulting in an increase in fluorescence and a shift in the emission maximum from 500 to 460 nm. 12-Jun-12

ESTIMATION OF GSH LEVELS :Piperlongumine treated higher concentrations showed significant decrease in GSH levels determined using ONEWAY-ANOVA.

6/12/201235ROS-mediated apoptotic signalling is associated with decreased cellular GSH levels and the loss of cellular red-ox balance. Decreased intracellular GSH can occur through ROS-induced GSH oxidation or GSH export from cells; the resultant GSH reduction would enhance further ROS production during oxidative challenge .

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ESTIMATION OF CATALASES ACTIVITY :Piperlongumine treated cells showed dose-dependent increased %H2O2(V/V) that indicates decreased catalases activity .

6/12/20123612-Jun-12ESTIMATION OF SUPEROXIDE DISMUTASE ACTIVITY:Piperlongumine treated cells showed dose dependent decrease in %O22-Inhibition comparison to control cells. Pre-treatment with Nac showed increased %O22-Inhibitory activity.(Standard reference)

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WST-1(2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt) that produces a water-soluble formazan dye upon reduction with a superoxide anion. The rate of the reduction with O2-is linearly related to the xanthine oxidase (XO) activity, and is inhibited by SOD.12-Jun-12

MEASUREMENT OF SUPEROXIDE ANION PRODUCTION:Piperlongumine treated cells showed dose dependent increase in superoxide anion production in comparison to control cells.

6/12/201238water soluble NBT is converted into a water insoluble blue formazan crystal by the action of superoxide anions.12-Jun-12

MATRIX METALLOPROTEINASE-2 INHIBITION:Piperlongumine showed dose dependent increase in MMP2 inhibition with respect to the relative fluorescence units in comparison to control cells.

6/12/201239Mca [Mca= (7-methoxycoumarin-4-yl)-acetyl; fluorescence is quenched by the Dpa (Dpa=N-3-(2, 4-dinitrophenyl)-L---diaminopropionyl] group until cleavage by MMPs at the Gly-Leu bond separates the two moieties.12-Jun-12

MATRIX METALLOPROTEINASE-9 INHIBITION:Piperlongumine showed dose dependent increase in MMP9 inhibition with respect to the relative fluorescence units in comparison to control cells.

6/12/201240MMP-9 inhibitors using a quenched fluorogenic peptide: OmniMMPTM fluorogenic substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 [Mca=(7-methoxycoumarin-4-yl)-acetyl; Dpa=N-3-(2,4-dinitrophenyl)-L---diaminopropionyl]. Mca fluorescence is quenched by the Dpa group until cleavage by MMPs at the Gly-Leu bond separates the two moieties.12-Jun-12

ESTIMATION OF CASPASE-3 ACTIVITY:Piperlongumine showed dose-dependent increase in Caspase-3 expression and was confirmed by addition of inhibitor.

6/12/201241The caspase 3 colorimetric assay is based on the hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) by caspase 3, resulting in the release of the p-nitroaniline (pNA) moiety. p-Nitroaniline has a high absorbance at 405 nm (mM= 10.5). The concentration of the pNA released from the substrate is calculated from the absorbance values at 405 nm or from a calibration 1curve prepared with defined pNA solutions. Caspase 3 Ac-DEVD-pNA Ac-DEVD + pNA 12-Jun-12In 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 & anti-apoptotic proteins and activation of caspase-3.

Further experimentation on Piperlongumine in animal models is essential to take this compound further for clinical use.

CONCLUSION:

6/12/201242PROPOSED MECHANISM OF PIPERLONGUMINE:

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6/12/201247ACKNOWLEDGEMENTS:Dr. Ahmed KamalDr. Sistla RamakrishnaMr. T.VenuDr.V.G.M.NaiduMr. Kulkarni NIPER FacultyIICT LabmatesNIPER Friends

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Thank you

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