Activation of caspase 3&9 by methanol extract of Gloriosa superba 

ABSTRACT

Mitochondrion is the key regulator in cellular energy homeostasis and plays a central role in determining cell apoptotic process; it is therefore regarded as a vital target for cancer chemotherapy. Cancer is obviously one of the most common areas where demand for alternative treatment is overwhelming. Many investigations revealed that bioactive compounds in natural products can act on mitochondria to trigger the permeabilization of the mitochondrial outer membrane and lead to the impairment of the mitochondria, including the alteration of electron transport, the loss of mitochondrial transmembrane potential, and the cytosolic release of apoptotic proteins such as Cytochrome C. Gloriosa superba is a medicinal plant used traditionally as anti-fungal, anti-bacteria and anti-carcinogenic agent. This study evaluates the modulatory effects of varying concentrations (10, 30, 50, 70 and 90µg/ml) and varying doses(50,100,200 and 400mg/kg) of methanol extract of Gloriosa superba on rat liver mitochondrial membrane permeability transition pore (mPT),as well as the release of caspases 9 and 3 which are the main regulators of mitochondria-mediated apoptosis.

Rats with approximately 100g weight were used for this experiment. Rat liver mitochondria were isolated by differential centrifugation, the mPT pore opening ,ATPase activity, lipid peroxidation, Cytochrome C release were assayed for at 540nm,660nm,532nm and 414nm respectively.

In the presence of calcium, there was induction of opening of the mPT pore which was reversed by spermine, a standard inhibitor. In the absence of calcium, methanol extract induced mPT pore opening in a concentration dependent manner. Mitochondrial ATPase activity was enhanced by the methanol extract with highest ATPase activity of 79% at 90µg/ml. The MEGS also inhibited Fe2+-induced lipid peroxidation with the highest inhibition at 50µg/ml. MEGS also induces Cytochrome C release at the highest concentration of 90µg/ml. 

The in-vivo analysis also indicated that the extract induced mPT pore opening of rat liver mitochondria in a dosage-dependent manner, with the highest induction at 400mg/kg. Oral administration of MEGS also indicated increase in the activation of caspases 9 and 3, the initiator and executioner of mitochondria-mediated apoptosis.

These findings suggest that the bioactive agents that induce pore opening are present in the methanol extract and thus may be used as drug candidates in situations where apoptosis up regulation is necessary.

TABLE OF CONTENTS

TITLE PAGE………………………………………………………………………………………I

CERTIFICATIONII

DEDICATIONIII

ACKNOWLEDGEMENTSIV

TABLE OF CONTENTSV

LIST OF FIGURESIX

LIST OF TABLES.XII

ABSTRACTXIII

CHAPTER ONEI

INTRODUCTION AND LITERATURE REVIEWI

1.0 INTRODUCTIONI

JUSTIFICATIONIVOBJECTIVES OF THE STUDYVLITERATURE REVIEWVI

1.3.0 MITOCHONDRIAVI

STRUCTURE OF THE MITOCHONDRIONVII

1.3.2 Energy Metabolism in MitochondriaXI

Heat ProductionXIV

1.3.4 Oxidative PhosphorylationXIV

1.4. MORPHOLOGY OF APOPTOSISXXII

1.4.1 The Significance of ApoptosisXXIII

1.4.2 Distinguishing Apoptosis from NecrosisXXIV

1.4.3 Mechanisms of ApoptosisXXVIII

1.4.4 CASPASES: The Initiators and Executioners of ApoptosisXXXII

1.4.5 PERFORIN/GRANZYME PATHWAYXXXV

1.4.6 REGULATION OF APOPTOSIS BY IAPSXXXVII

1.4.7 INHIBITION OF APOPTOSISXL

1.4.8 ASSAYS FOR APOPTOSISXLI

1.4.9 APOPTOSIS IN DISEASE PATHOLOGYXLII

1.5 MITOCHONDRIA IN APOPTOSISXLVI

1.5.1 MITOCHONDRIAL MEMBRANE PERMEABILITY TRANSITION (mPT)LII

1.6 MEDICINAL PLANTSLX

1.6.1 Gloriosa superbaLXI

CHAPTER TWOLXVII

2.1  PLANT MATERIALLXVII

2.1.1  Collection of plant materialLXVII

2.1.2 Preparation of extractLXVIII

2.1.3 Experimental animalsLXVIII

2.2 PREPARATION OF LOW IONIC STRENGTH LIVER MITOCHONDRIALXVIII

2.2.1 REAGENTSLXVIII

2.2.2 PROCEDURELXIX

2.3 PROTEIN DETERMINATIONLXX

2.3.1 PRINCIPLELXX

2.3.2 REAGENTSLXXI

2.3.3 PREPARATION OF FOLIN-CIOCALTEAU REAGENTLXXI

2.3.4 STANDARD PROTEIN SOLUTIONLXXII

2.3.5 PROCEDURELXXII

2.3.6 SAMPLE PREPARATIONSLXXII

2.4 ASSESSMENT OF MITOCHONDRIAL MEMBRANE PERMEABILITY TRANSITION IN RAT LIVER MITOCHONDRIALXXV

2.4.1 PRINCIPLELXXV

2.4.2 REAGENTSLXXV

2.4.3 PROCEDURELXXVI

2.5 DETERMINATION OF MITOCHONDRIAL ATPase ACTIVITYLXXVIII

2.5.1  PRINCIPLELXXVIII

2.5.2 REAGENTSLXXVIII

2.5.3 PROCEDURELXXIX

2.5.4 DETERMINATION OF INORGANIC PHOSPHATELXXX

2.6 DETERMINATION OF LIPID PEROXIDATIONLXXXIII

2.6.1 PRINCIPLELXXXIII

2.6.2 REAGENTSLXXXIV

2.6.3 PROCEDURELXXXIV

CHAPTER THREELXXXVI

3.1 EVALUATION OF THE EFFECT OF Ca2+ AND SPERMINE ON RAT LIVER MITOCHONDRIAL MEMBRANE PERMEABILITY TRANSITION PORE.LXXXVI

3.1.1 INTRODUCTIONLXXXVI

3.1.2 PROCEDURELXXXVII

3.1.3 RESULTLXXXVII

3.1.4 CONCLUSIONLXXXVIII

3.2 EVALUATION OF THE EFFECT OF VARYING CONCENTRATIONS OF METHANOL EXTRACT OF GLORIOSA SUPERBA ON RAT LIVER MITOCHONDRIA MEMBRANE PERMEABILITY TRANSITION PORE IN THE ABSENCE AND PRESENCE OF CALCIUM.LXXXIX

3.2.1 INTRODUCTIONLXXXIX

3.2.2 PROCEDUREXC

3.2.3 RESULTSXC

3.2.4 CONCLUSIONXCI

3.3 EVALUATION OF EFFECTS OF VARYING CONCENTRATIONS OF METHANOL EXTRACT OF Gloriosa superba ON RAT LIVER MITOCHONDRIA ATPase ACTIVITY.XCIII

3.3.1 INTRODUCTIONXCIII

3.3.2 PROCEDUREXCIV

3.3.3 RESULTXCIV

3.3.4 CONCLUSIONXCIV

3.4 EVALUATION OF EFFECTS OF VARYING CONCENTRATIONS OF METHANOL EXTRACT OF Gloriosa superba ON RAT LIVER MITOCHONDRIA CYTOCROME C RELEASE.XCVI

3.4.1 INTRODUCTIONXCVI

3.4.2 PROCEDUREXCVI

3.4.3 RESULTXCVII

3.4.4 CONCLUSIONXCVII

3.5 EVALUATION OF THE EFFECT OF VARYING CONCENTRATIONS OF METHANOL EXTRACT OF Gloriosa superba ON LIPID PEROXIDATION IN NORMAL RAT LIVER MITOCHONDRIA.XCIX

3.5.1 INTRODUCTIONXCIX

3.5.2 PROCEDUREXCIX

3.5.3 RESULTC

3.5.4 CONCLUSIONC

EXPERIMENT 6CII

3.6 EVALUATION OF VARYING DOSES OF THE METHANOL EXTRACT OF Gloriosa superba ON RAT LIVER MITOCHONDRIA MEMBRANE PERMEABILITY TRANSITION PORECII

3.6.1 INTRODUCTIONCII

3.6.2 PROCEDURECII

3.6.3 RESULTSCIII

3.6.4 CONCLUSIONCIII

EXPERIMENT 7CIV

3.7 INFLUENCE OF METHANOL EXTRACT OF Gloriosa superba ON CASPSES 9 AND 3 ACTIVATION USING ELISA TECHNIQUE.CIV

3.5.1 INTRODUCTIONCIV

3.5.2 PROCEDURECIV

3.5.3 RESULTSCV

3.5.4 CONCLUSIONCV

CHAPTER 4CVIII

4.1 DISCUSSIONCVIII

4.2 CONCLUSIONCX

REFERENCES.CXI

LIST OF FIGURES

Figure 1.3.1: Structure of the Mitochondrion

Figure 1.3.2: Electron transport sequence 

Figure 1.3.4:Oxidative phosphorylation

Figure 1.4: Hallmarks of the apoptotic and necrotic cell death process

Figure 1.4.3: Intrinsic mitochondria-mediated pathway 

Figure 1.4.5: Apoptotic pathways 

Figure 1.4.6: Mammalian IAP family members 

Figure 1.5: Displacement of IAPs from caspases by Smac/Diablo 

Figure 1.5.1: Mitochondria: promising targets for cancer chemotherapy

Figure 1.6: Gloriosa superba leaf

Figure1.6.1.2: Colchicine

Figure 2.3: Standard protein curve

Figure 2.5.4: Standard phosphate curve

Figure 2.6: MDA reaction in lipid peroxidation assay

Figure 3.1: Calcium-induced mitochondrial membrane permeability transition pore opening in normal rat liver mitochondria and its reversal by spermine

Figure 3.2:Effects of varying concentrations of MEGS on rat liver mitochondrial Membrane permeability transition pore in the absence of calcium

Figure 3.3: Effects of varying concentrations of MEGS on mitochondrial ATPase activity

Figure 3.4: Effects of varying concentrations of MEGS on Cytochrome C release. 

Figure 3.5: Effects of varying concentrations of MEGS of on lipid peroxidation.

Figure 3.6: Effects of varying doses of MEGS on rat liver mitochondrial membrane permeabilitytransition pore.

Figure 3.7:Effects of oral administration of MEGS on caspase 9 activation

Figure3.8:Effects of oral administration of MEGS on caspase 3 activation.

LIST OF TABLES.

Table 1.1: Inhibitors of oxidative phosphorylation 

Table 1.2: Subfamily of Caspases

Table 2.1: Protocol for Protein Estimation

Table 2.2: Protocol for Mitochondrial swelling assay