TABLE OF CONTENTS
TABLE OF CONTENTS ............................................................................................. iii
LIST OF TABLES ....................................................................................................... vi
LIST OF FIGURES .................................................................................................... vii
LIST OF APPENDICES ............................................................................................ viii
ACKNOWLEDGEMENT .............................................................................................x
ABSTRACT ................................................................................................................. xi
INTRODUCTION..............................................................................................................1
Background of the Study ...............................................................................................1
Significance of the Study ...............................................................................................2
Research Question .........................................................................................................3
General Objective ..........................................................................................................3
Specific Objectives ........................................................................................................3
Conceptual Framework ..................................................................................................4
REVIEW OF RELATED LITERATURE ......................................................................5
The Significance of Chemotherapy ...............................................................................5
Factors Affecting the Overall Health Outcome After Chemotherapy ...........................6
The Adverse Effects of Doxorubicin: A Widely Used Chemotherapy Drug ................8
Chemoprotective Phytochemicals Found in Mango Peel Extracts ................................9
Cytoprotective Drugs Against Doxorubicin Toxicity ..................................................10
HK-2 Human Proximal Tubular Cell Lines .................................................................11
Cell Confluency of HK-2 Cells for Experimental Treatment Conditions ...................12
MTT Assay Used for Cell Viability.............................................................................13
METHODOLOGY ..........................................................................................................15
Study Design ................................................................................................................15
Study Setting ................................................................................................................15
Population and Sampling Technique ..........................................................................15
iii
Inclusion Criteria .................................................................................................................. 16
Exclusion Criteria ........................................................................................................16
Sampling Procedure and Comparison Groups .............................................................16
Sample Size Computation ............................................................................................17
Operational Definition of Terms ..................................................................................17
Data Collection Procedure ...........................................................................................18
a.) Extraction of Ripe Mango Peel ............................................................................19
b.) Mango Peel Extract Dilution ...............................................................................21
c.) HK-2 Human Proximal Kidney Cell Lines Culture.............................................21
d.) IC50 of MPE and Doxorubicin Treatment in HK-2 Human Proximal Kidney ...22
e.) Experimental Set-up for MPE Treatment ............................................................22
f.) Percent Cell Viability Measurement via MTT Assay ..........................................23
g.) Data Processing and Analysis..............................................................................24
h.) Scope and Limitations .........................................................................................25
i.) Ethical Consideration ............................................................................................25
RESULTS .........................................................................................................................26
Mango Peel Extract for Chemoprotection Against Doxorubicin-Induced
Nephrotoxicity ...................................................................................................................26
IC50 of MPE and Doxorubicin Treatment in HK-2 Cells ...........................................26
Cell Morphology of HK-2 Cells After Each Treatment Conditions ............................26
Chemoprotective Effects of MPE Against Doxorubicin Toxicity in HK-2 Cells .......28
a.) Differences in Percent Cell Viability Among the Four Treatment Groups .........28
b.) Differences in Percent Cell Viability Between and Among Concentrations and
Treatment Groups .....................................................................................................29
c.) Differences in % Cell Viability Between Different Concentrations of MPE ......32
DISCUSSION ...................................................................................................................33
Cell Morphology of HK-2 Cells After DOX-Induced Nephrotoxicity and Subsequent
Treatment with MPE .....................................................................................................33
iv
Chemoprotective Property of MPE Against Doxorubicin Toxicity in HK-2 Cells ......34
CONCLUSION AND RECOMMENDATIONS ...........................................................36
Conclusion ...................................................................................................................36
Recommendations ........................................................................................................37
REFERENCES .................................................................................................................39
v
LIST OF TABLE/S
Table
Page
1 PotePotential cytoprotective products against DOX toxicity………………………
10
vi
LIST OF FIGURES
Figure
Page
1
Conceptual framework of Doxorubicin-induced nephrotoxicity and potential
chemoprotective mechanism of mango peel extract (MPE)................................ 4
2
Flowchart of the procedure for data collection…………………………………
3
(a) Air-dried mango peels after 4 weeks and (b) were pulverized using a blender,
and were then (3) strained to separate the large pieces from the fine powdered
form of the mango peels……………………………………………………….. 19
4
(a) Preparation of the mango peel extract in methanol before (b) rotary
evaporation to gently separate the extraction solvents used, that is, methanol
and ethyl acetate……………………………………………………………….. 20
5
Experimental set-up for DOX and MPE treatment in a 96-well plate for 48
hours. (V= vehicle; M= MPE; D= DOX; DM= DOX + MPE; B= blank: 2.5 =
2.5 ppm, 5 = 5 ppm, 10 = 10 ppm)...................................................................... 23
6
HK-2 human proximal kidney cells after 48 hrs (a) in a normal culture media
(vehicle; 2.5 ppm) showing adhering polygonal cells; (b) in a culture media
treated with 10 ppm MPE; (c) exposed to 2.1 ppm of DOX with circular cells
detached from adjacent cells with 38.6% cell viability, and; (d) exposed to 2.1
ppm of DOX treated with 10 ppm of MPE with _% cell viability. Adhering
polygonal cells are observed in cells treated with quercetin and MPE………… 27
7
Two-way ANOVA with Tukey multiple comparison (post-hoc analysis) results
in % cell viability of HK-2 cells treated with 2.5 ppm, 5 ppm and 10 ppm vehicle
and MPE, and HK-2 cells subjected to DOX-induced toxicity at 2.1 ppm and
treated with 2.5 ppm, 5 ppm, and 10 ppm MPE after 48 hours. (* = P
<0.05).................................................................................................................. 30
8
One-way ANOVA with Tukey multiple comparison (post-hoc analysis) results
in percent cell viability of HK-2 cells subjected to DOX-induced toxicity at 2.1
ppm treated with (a) 2.5 ppm, (b) 5 ppm, and (c) 10 ppm of vehicle and MPE
after 48 hours.
(ns = not significant (P > 0.05); * = P
<0.05))................................................................................................................. 31
9
One-way ANOVA with Tukey multiple comparison (post-hoc analysis) results
in % cell viability of HK-2 cells subjected to DOX-induced toxicity at 2.1 ppm
treated with 2.5 ppm, 5 ppm and 10 ppm MPE after 48 hours. (ns = not
significant; P > 0.05)........................................................................................... 32
vii
18
LIST OF APPENDICES
Appendix
Page
1
Calculation for % inhibition of MPE and DOX………………………………... 45
1
Table 1. Percentage inhibition of M. indica crude extract against HK-2 cells… 45
1
Table 2. Percentage inhibition of doxorubicin against HK-2 cells…………….. 45
2
Calculation of IC50 of MPE and DOX………………………………………… 46
2
Table 1. Absorbance values provided by the microplate reader during the
MTT assay of HK-2 cells treated with MPE and DOX………………………... 46
2
Figure 1. Linear graph obtained from generating the IC50 of MPE…………… 46
2
Table 2. Calculated IC50 values of DOX in HK-2 cells………………………... 47
2
Table 3. Percent cell viability of MPE on DOX-induced nephrotoxicity in HK2 cells…………………………………………………………………………… 47
3
Table 1. One- way ANOVA to test for the interaction of the four treatment
groups at 2.5 ppm………………………………………………………………. 48
3
Table 2. Tukey multiple comparison to test for the differences of % cell
viability among the four treatment groups at 2.5 ppm…………………………. 49
3
Table 3. One- way ANOVA to test for the interaction of the four treatment
groups at 5 ppm………………………………………………………………… 50
3
Table 4. Tuley multiple comparison to test for the differences of % cell
viability among the four treatment groups at 5 ppm……………………………. 51
3
Table 5. One- way ANOVA to test for the interaction of the four treatment
groups at 10 ppm………………………………………………………………. 52
3
Table 6. Tukey multiple comparison to test for the differences of % cell
viability among the four treatment groups at 10 ppm………………………….. 53
4
Table 1. One- way ANOVA to test for the interaction of the four treatment
groups at 2.5 ppm, 5 ppm and 10 ppm…………………………………………. 54
viii
4
Table 2. Tukey multiple comparison to test for the differences of % cell
viability among the four treatment groups at 2.5 ppm, 5 ppm and 10 ppm……. 55
5
Table 1. One- way ANOVA to test for the interaction of % cell viability of
2.5 ppm, 5 ppm and 10 ppm MPE ……………………………………………..
59
5
Table 2. Tukey multiple comparison to test for the interaction of % cell
viability of 2.5 ppm, 5 ppm and 10 ppm MPE…………………………………. 61
6
Table 1. Gantt Chart of Activities……………………………………………...
62
7
Table 1: Research budget for mango peel extraction, and cell culture and
treatment………………………………………………………………………..
63
8
Transmittal Letters……………………………………………………………... 64
9
Curriculum Vitae……………………………………………………………….. 71
10
Good Clinical Practice Certificates…………………………………………….. 80
ix
ACKNOWLEDGEMENT
We dedicate this thesis to the following people who never lose faith in us:
1. To our parents, who served as our lighthouse throughout this journey, we thank
them for their perpetual patience and understanding,
2. To Dr. Chamberlain Agtuca for constantly checking up on our progress, for always
accommodating our late night questions and consultations, and for his valuable
inputs on this study,
3. To our panel members, Dr. Helen Madamba, Dr. Maria Lucila Ilano, and Dr. Dexter
Dimaano for their significant suggestions, comments and corrections during the
proposal hearing and oral defense which helped shape the outcome of the study as
well as the manuscript,
4. To Dr. Paul Geraldino and Dr. Jonie Yee of the Department of Biology in the
University of San Carlos, for their generosity and for accommodating us in their
respective laboratories as we went through the entire process of this research,
5. To Mr. Norman Quilantang for assisting us in cell culture and MTT assay, and
8. To our Almighty God, who is always good, always sustaining and constant, we
offer this success for His greater glory.
x
ABSTRACT
Introduction: Cancer chemotherapy treatment has long been an important part of cancer
management. However, chemotherapy is met with setbacks due to its
nonselective nature which can also induce toxic effects to normal cells. On the
other hand, mango peel extracts (MPE) contain biologically active metabolites
such as flavonoids, carotenoids and quercetin which are known for its
therapeutic potential. Thus, this study aimed to determine the protective effects
of MPE against doxorubicin (DOX) cytotoxicity in HK-2 human proximal
kidney cell lines.
Methods:
The study utilized a true experimental study design. To induce nephrotoxicity
on HK-2 cells, 2.1 ppm of DOX was added to the culture media. Peels from
ripe mangoes were initially dried and extracted. The extracts were then serially
diluted to 2.5 ppm, 5 ppm and 10 ppm which were used to treat on DOXinduced HK-2 human proximal kidney cells for 48 hours. Both one-way and
two-way analysis of variance (ANOVA) with Tukey multiple comparison as
post-hoc analysis were utilized for data analysis.
Results:
HK-2 cells exposed to 2.1 ppm DOX treated with 2.5 ppm, 5 ppm, and 10 ppm
MPE had an average % cell viability of 46.9%, 44%, and 45.9% after 48 hours,
respectively. These are significantly higher compared to cells treated with 2.1
ppm DOX alone which was only at 36.5%. Thus, MPE was able to protect HK2 cells from DOX-induced nephrotoxicity by significantly increasing % cell
viability by 10.4% at 2.5 ppm (P < 0.0001), 7.5% at 5 ppm (P < 0.0001), and
9.4% at 10 ppm (P < 0.0001) after 48 hours. Moreover, there is no significant
difference (P > 0.05) between each treatment concentration of MPE suggesting
that its potential chemoprotective property is not dose-dependent.
Conclusion:
MPE was able to ameliorate the nephrotoxic effects of DOX. Additionally, it
has been shown that the chemoprotective property of MPE was not dosedependent at a concentration range of 2.5 - 10 ppm. These results feature the
promising chemoprotective properties of MPE against DOX-induced
nephrotoxicity in HK-2 human proximal kidney cell lines.
Keywords:
chemoprotection, mango peel extracts, proximal kidney cells
xi