Cytotoxicity sceening of extracts from combretum quadrangulare on MCF-7 breast cancer cell line.pdf

Thesis’s title: Cytotoxicity screening of extracts from Contbretunt quadrangulare on MCF-7 breast cancer cell line.. extracts from Combretum quadrangulare on MCF-7 breast cancer cell li

NGUYEN TAT THANH UNIVERSITY

NGUYEN TAT THANH

True learning, true practice, true success, true future

GRADUATION THESIS

Phuong Thi Nguyen, MSc

Ho Chi Minh City, 2019

NGUYEN TAT THANH

UNIVERSITYTrue learning, true practice, true success, true future

GRADUATION THESIS

Student’s name

Student ID

Major Supervisors

: Thu Thi My Tran

: 1411534235: Biotechnology: Hung Huu Nguyen, PhD

Phuong Thi Nguyen, MSc

Ho Chi Minh City, 2019

NGUYEN TAT THANH UNIVERSITY

FACULTY OF BIOTECHNOLOGY

SOCIALIST REPUBLIC OF VIETNAM Independence - Liberty - Happiness

Full name: Thi My Thu Tran Student ID: 1411534235

1 Thesis’s title:

Cytotoxicity screening of extracts from Contbretunt quadrangulare on MCF-7

breast cancer cell line.

2 Objectives

- Screening for extracts or fractions from Combretum quadrangulare based on

cytotoxic activity onMCF-7 breastcancer cellsusing MTT assay

- Study on other bioactivity of the best cytotoxic fraction as antibacterial andantioxidant ability

3 Contents

- Determination of time culture forMTT assay

- Screening extracts or fractions ateach extraction phase ofCombretum.

morphology and IC50 values

- Determine the fraction with the best cytotoxicity on MCF-7 cells through IC50values to isolate bioactive compounds

- Determine antibacterial and antioxidant activity of the best cytotoxic fraction

4 Execution time: from 12/2018 to 09/2019

5 Supervisor: Huu Hung Nguyen, PhD

Co-supervisor: PhuongThi Nguyen, MSc

Contents and requirements of this thesis were adopted by subject

HCM city, 2019

With sincere feelings, I would like to express my gratitude to my supervisors

Hung Huu Nguyen, PhD and Phuong Thi Nguyen, MSc Faculty of Biotechnology,

Nguyen Tat Thanh University (NTTU) in particular and all of brothers, sisters, friends

in the stem cell laboratory who have devotedly helped me to complete this graduation

thesis I would also like to thank the teachers in NTTU who have taught me a lot of useful knowledge to carry out the thesis and have a solid luggage for my future career

Becauseof the limitations of knowledge and reasoning abilityof myself,there are many

shortcomings and limitations, I hope to receive the instruction and contribution of

teachers so that my thesis willbe improved

Thu Thi My Tran

Faculty of BiotechnologyNguyen Tat Thanh University

1

TABLE OF CONTENTS

ACKNOWLEDGEMENTS i

TABLE OF CONTENTS ii

ABSTRACT iv

LIST OF FIGURES V LIST OF TABLES vi

LIST OF ACRONYMS vii

INTRODUCTION viii

CHAPTER 1 LITERATURE REVIEW 10

1.1 Cancer 10

1.1.1 Cancer overview 10

1.1.2 Breast cancer 11

1.2 Combretum quadrangulareoverview 13

1.2.1 Characteristics 13

1.2.2 Bioactivity ofCombretum quadrangulare extracts and compounds 14

CHAPTER 2 CONTENTS AND METHODS 18

2.1 Place of administration 18

2.2 Contents 18

2.3 Methods 18

2.3.1 Cells preparation 18

2.3.2 MTT method 20

2.3.3 Agar Well Diffusion 23

CHAPTER 3 RESULTS AND DISCUSSION 25

3.1 Determination of time culture for MTT assay 25

3.2 Screening of the extracts based on cytotoxicity ability 26

3.2.1 Screening ofc quadrangualre Crude extracts 26

3.2.2 Screening of Liquid-Liquidextracts 27

3.2.3 Screening of cytotoxicity of P1-P14 and PAX1 fractions on MCF-7 cells 29

3.2.4 Other Bio-activityofP12 fraction 33

CONCLUSIONS AND RECOMMENDATIONS 35

REFERENCES 36

APPENDICES 37

iii

extracts from Combretum quadrangulare on MCF-7 breast cancer cell line" is carried out from 12/2018 to 09/2019 in Cancer cell and Stem cell laboratory, Faculty of

Biotechnology and Hi-tech Institute, Nguyen Tat Thanh University Withthe goal: To

screening the cytotoxicity possibility of the extracts/fractions from Combretum

purpose: To determine the fraction from Combretum quadrangulare that have the best

effect on MCF-7 cellsfor the useofbioactive compounds isolation

The topic has two main contents: (1) Screening for extracts or fractions from

using MTT assay (2) Study on other bioactivity of the best cytotoxic fraction asantibacterial and antioxidant ability

With the obtained results: (1) EtOH extract from the leafwas determined as thebest cytotoxicity on MCF-7 cells; H:Ea was determined as best solvent for EtOH leaf extract; from H: Ea extract, fraction P12 was capable of causing thehighestMCF-7 cell

toxicity (2) Fraction P12 had quite good antibacterial effecton Staphylococcus aureus,

ability with IC50 was 96.3 ± 1.8 pg/ml

Figure 3.2 Cytotoxic activity on MCF-7 of Doxorubicin, EtOH extract from leaf,root,

seed The celldeathrateis expressed as the mean ofthree repetitions and SD standard

deviation 26

Figure 3.3 Breast cancer cytotoxic activity MCF-7 of the fraction Bu, Ea, H, H:Ea and

Doxorubicin The cell deathrate isexpressedas the mean of three repetitions and SDstandard deviation 28

Figure 3.4 MCF-7 cytotoxic activity ofthe fraction Pl- P14, PAX1 and Doxorubicin

The cell death rate is expressed as the mean of three repetitions and SDstandard

deviation 30

Figure 3.5 The antibacterial activity ofP12 fraction and 10 pg/mL Kanamycin (Kan)

were implemented against two Gram positive bacteria(Ổ cereus-BC and 5

aureus-SA)and two Gram negative bacteria (P aeniginosa-PA and E coli-EC) 33

Figure 3.6 DPPH scavenging activity of (A) Ascorbic acid (Vitamin C) and (B) P12

fraction from c quadrangulare Inhibition (%) is expressed as the mean of three

repetitions and SD standarddeviation 34

V

LIST OF TABLES

Table 3.1 Changes in MCF-7 morphology aftertreating EtOH extracts from leaf, root,

seedat a concentration of 50 pg/ml at 24, 48 and 72 hours of culture 26

Table 3.2 IC50 value of EtOH leaf, root, and seed extract ofc quadrangulareon

Table 3.3 Changes in MCF-7 morphology aftertreating Bu, H: Ea, Ea and Hextracts at

a concentration of 50 pg/ml at 24, 48 and 72 hours of culture 28

Table 3.4 IC50 value of H, H:Ea, Ea, Bu extract of c quadrangulare on MCF-7 29

Table 3.5 Changes in MCF-7 morphology after treating P1-P14, PAX1 fractions at a

concentrationof50 pg/ml at 24,48 and 72 hours of culture 30

Table 3.6 IC50 value of Pl-P14 and PAX1 fractions of Cotnbretum quadrangulare on

breast cancer cell MCF-7 32

LIST OF ACRONYMS

WHO World Health Organization

DMSO Dimethyl sunfoxide

FBS FetalBovine Serum

PBS Phosphate Buffered Saline

EDTA Ethylendiamin Tetraacetic Acid

MHA MuellerHinton Agar

DPPH 2,2-Diphenul-1 -picrylhydrazyl

Vil

2030 the number of cancer cases in the world will increase manytimes over the current

figure andthe number ofcancer deaths willalso double - more than 13.2 million people

in 2030 In Vietnam, according to the Cancer Prevention Research Institute, about94,000 people die from cancereach year, the cancer mortality rate in Vietnamranks 78

out of 171 countries being regulatedlook up The worrying issuenow is thatthe number

of people with cancerin Vietnam tends to increase rapidly In 2000,Vietnam had about

69,000 new cases of cancer, in 2015 up to 150,000 new cases It is estimated that by

2020 the number of new cancers in Vietnamwillbe approximately 200,000

In particular,breast cancer is themostcommon cancer in women and is the second

most common cancer in the world Worldwide estimates have more than 508,000women died in 2011 due to breast cancer In Vietnam, the proportion of women with breast cancer has increased rapidly in recent yearswith more than 10,000 new patients

each year and 4,500 deaths

With the above situation, the research on cancer and cancer treatment is a very

concern in the whole world There have been many organizations as well as cancer

research institutes inthe world established with thepurpose of in-depth research as well

as finding treatments for cancer Currentlythe most optimal breast cancer treatment is surgical removal, chemotherapyand radiotherapy However, drugs usedto treat cancercurrently are mostly synthetic drugs causing drug resistance Therefore, it is necessary

to find new drugs that can killcancer cells in cancer treatment

In recent years, research on the active compounds that areextracted fromplants is

growing Among them, some compounds belong to alkaloid groups, terpenoids,phenolic compounds, extracted saponins are known tobe compounds that cankill cancer

cells On the other hand, Combretum quadrangulare is a quite popular plant in the

southern plains of Vietnam buthasmany usessuch as antibacterial, anti-HIV, poisoningability cancer cell Extracts as well ascompounds extractedfrom a number of parts such

as roots, flowersand leavesof Combretum quadrangulare in Vietnam aswell as in theworld have been shown to have toxic effects on many types ofcancer cells, especiallyfor breast cancer Although many compounds have been studied and extracted from

high biological activity

In addition, the research team at the Stem Cells and Cancer Laboratory in 2017has demonstrated the extracts fromthe leaves of Combretumquadrangulare,which has

a strong toxic effect on Hep-G2 liver cancer and K562 blood cancer cells With these prerequisite results, the cytotoxicity screening procedure based on MTT assay was

completed to isolate the active fraction with a strong biological activity on cancer cellline

Forthepurpose of screening of extracts from Combretum quadrangulare based on

MCF-7 breast cancercytotoxicity to find the fraction with the best toxicity on MCF-7cells, the topic ’’Cytotoxicity screening of the extracts from Contbretuni

quadrangulare on MCF-7 breast cancer cell line” was implemented

IX

Chapter 1 Literature review

1.1 Cancer

1.1.1 Cancer overview

Cancer was considered as a major burden of theworld Every year,tens of millions

of people are diagnosedwith cancer worldwide andmore thanhalfofpatients died from

cancer In many countries, cancer is the second most common cause of death after

cardiovascular disease Based on GLOBOCAN database, there wereabout 10.8 million

newcasesof cancer (excluding skin cancer) overtheworldin2002, of which5.8 million

ofpeople (53.4%) were male and 5 million (46.6%) were female Nearly 45% of new

cases were diagnosed in Asia, 26% in Europe, 15% in North America, 7% in Latin America and 6% in Africa In females, the top common cancer is breast cancer (1.1 million new cases every year), followed by cervix cancer (493,100 cases) and colon

cancer (472,743 cases) In males, the three most common cancer are lung cancer

(965,446 cases), prostate cancer (679,060cases) and stomachcancer (603,003 cases)1

In 2002, the numberof cancer deathsworldwide were6.7 millioncases, of which

3.8 million cases were male and2.9 million cases were female Thetotalnumberoflung

cancer deaths were 1,179,074 cases, of which 848,321 cases were male and 330,753cases were female Next, stomach cancer resultedin a totalof 699,803 deaths, including 445,691 cases in men and 254,112 cases in women Liver cancer is the third cause ofdeath from cancer all over the world For women, the top three causes ofdeath from cancer are breast cancer(411,093 deaths), lung cancer (330,753 deaths) and uterine and

cervical cancer (273,449 cases) Lung cancer (848,321 cases), stomach cancer (445,691

cases) and livercancer (416,926 cases) are the top three causes of cancer for men

In 2018,over9.6 millionpeopleworldwide wereestimatedtodie ofcancer About

1.16 trillion USD is the total annual economic cost forcancer in 2010 and only30-50%

of cancers can be prevented The total number of people with cancer in all countries inthe world in 2016 is shownin Figure 1.1

Chapter 1 Literature review

tn Data

Number of people with cancer, 2016

Total number of people suffering from any type of cancer at a given time This IS measured across both sexes, and all ages

0 50.000 500,000 2 million >8 million

No data 10,000 100,000 1 million 4 million

Figure 1.1 Map showingthe numberof cancer cases by countryand region in the

world in 2016 (https://ourworldindata.org/cancer)

In particular, cancer was found in most countries, the number of people with cancerwas represented by the colorfrom boldto light, corresponding to the number ofpeoplewith cancer from high to low, countries with the ratio of the highest cancer patients are

China (8.36 million), followed by the United States (6.8 million), Japan (2.47 million), India (2.04 million) and Germany (1.91 million) people In Vietnam, the number of

new cases ofcancerincreased rapidly from 68,000 in 2000 to 126,000 in 2010 and was

expected to surpass 190,000 cases in 2020 Each year, about 115,000 people died from cancer, equivalent to 315 people/day WHO ranked Vietnam in top 2 ofthe top 50

countries in terms of new cases of cancermap2

1.1.2 Breast cancer

Breastcancer is themostcommon type of cancerinwomen and is the second most common cancer in both developed and developing countries Worldwide estimates of

more than 508,000women died in 2011 from breast(WHO, 2013), affecting 2.1 million

women a yearandalsocausingnumber of mostcancer-relateddeaths in women In 2018,

627,000womendiedof breast cancer, equivalent to 15% ofallcancer deathsin women

11

Chapter 1 Literature review

According to statistics, there were more than 2 million cases of new breast cancer in

2018

The incidence was very different worldwide from 19.3 outof 100,000 women in East Africa to 89.7 out of 100,000 women in Western Europe In most developing

countries, the incidence was less than 40 per 100,000 people The number of people

suffering from cancers in the world in 2016 is shown in the Figure 1.2

Number of people with cancer by type, World, 2016

Total number of people suffering from cancer at any given time, differentiated by cancer type This IS measured across both sexes and all ages

Breast cancer Colon & rectum cancer Prostate cancer

8 15 million 6.32 million

5.7 million Tracheal, bronchus, and lung cancer 2.84 million

Stomach cancer 2 2 million Uterine cancer 196 million Cervical cancer 1.94 million Bladder cancer 1.77 million Lip & oral cancer 1.39 million Kidney cancer 1.29 million Thyroid cancer 1.23 million Liver cancer 1.03 million Non-melanoma skin cancer 857,216 Ovarian cancer 785.602 Brain & nervous system cancer 781.185

Larynx cancer 637,720 Esophageal cancer • m 556,241 Pancreatic cancer 363,570 Testicular cancer 339,826 Nasopharynx cancer 332.016 Gallbladder & biliary tract cancer 169.019

0 1 million 3 million 5 million Source: IHME Global Burden of Disease

women with breast cancer has increased rapidly in recentyears with more than 10,000new patients each year and 4,500 deaths (NCBI, 2017)

Breast cancer survival rates vary widely worldwide, 80% or more of survivors ofthe total number of cases in North America, Sweden and Japan; in middle-income

countriesabout60%and less than40%forlow-incomecountries3 Becausethe situation

Chapter 1 Literature review

of cancer worldwide is alarming, study on cancer diagnosis and treatment is veryimportant

The big issue of cancer treatment is drug resistant cancer cells, so it is necessary

to find new compounds that can kill cancer cells effectively The hot issue ofcancer

treatmenttoday is that cancercells are resistant to the disease, so it is necessary to find

compounds that can kill cancer cells Among those compounds, the compounds

extracted from plants are the most popular Specifically, in this study, we performed ahigh screening ofnatural compounds from Combretum quadrangulare that had toxic

effects on breast cancer cells

1.2 Contbretuni quadrangulare overview

1.2.1 Characteristics

The Combretaceae family consists of 600 species of plants, in the range of 18-20 genera Plants of this family are found in tropical and subtropical regions, mainly in

Africa and India Two largest genera are Combretum and Terminalia, consisting of

about 370 and 200species, respectively Combretum quadrangulare (C quadrangulare)

is the most noted species of Combretum genus

Figure 1.3 The gourdCombretum quadrangulare4.

Combretum quadrangulare is a small tree of the Combretaceae family Grows wild

or grown very popular in Vietnam, Cambodia, Laos, Myanmarand Thailand Height is

5-10m: young branches have a narrow quadrilateral or quadrilateral shape Leaves are

13

Chapter 1 Literature review

simple, symmetrical,elliptical or obovate Flowers aresmall, yellow-white Dried fruits,

thin quadrilateral, reddish-brown seeds withelliptical shape4 comers, fruit season of c

1.2.2 Bioactivity of Combretum quadrangulare extracts and compounds

Current studies provided an assessment of the chemistry and pharmacology of

the most important plant species in the traditional medicine and established medical

system Combretum quadrangulare parts contain abundant source of compounds like

terpenoids, flavonoids, steroids and rude extracts also exhibit bio-activities such as

antimicrobial, anti-HIV, cytotoxic and hepato protective activities5

1.2.2.1 Antimicrobial activity

The methanol extract of c quadrangulare showed the antimicrobial activity

against gram positive cocci as methicillin-resistant s aureus and s aureuswith a MIC

value of 1212 pg/ml and 606,25 pg/ml The extract was also proved the antibacterial

activity against non-fermentable gram negative like p aeruginosa ATCC 27853 and

gram negative bacilli E coli with MIC value of 9,700 pg/ml6

Somanabandhu and colleagues reported thatthe ethanol and etherextract of dried

root bark or the dried seed of c quadrangulare had effect on earthworms when tested

epimastigote of Trypanosoma cruzi, the causative agent ofChagas disease; in which

acetone extract has the strongest effect7

1.2.2.2 Anti-HIV activity

The aqueous and ethanol extracts of c quadrangulare were screened for theirinhibitory activity against HIV-1 integrase (IN) - an enzyme essential for viral

replication The researchers noted significant inhibitory activity against the tested

enzyme of aqueous and ethanol extract having IC50 values of 2.5 and 2.9 pg/mL,respectively8

Chapter 1 Literature review

1.2.2.3 Cytotoxic activity

Cycloartane tritererpenoids and flavonoids isolated from methanol extract are

capable of cytotoxicity against elevated liver metastases on 26-L5 (in mice) carcinoma

cells Terpenoids of methyl quadrangutarate B and D were tested and resulted in strong

cell toxicity with ED50 values of9.54 and 5.42 pM, whileall flavonoids showed strong toxicity with low ED50 values, less than 6 pM9

From methanol extract of Combretum quadrangulare leaf, there are 15 new cycloartane triterpene as A-methyl quadrilateral A and N, p, 24-epiquadrangularate

methyl c, quadrilateral E, 23-deoxojessic acid were isolated The structure of new

compounds is demonstrated on the spectrum analysis and chemical conversion All

isolated compounds weretested fortoxicity to epithelial cells and mouse colon cells6

1.2.2.4 Cancer resistance

Currently there are 97 extracted compounds ofc quadrangulare are capable of

causing cancer cell toxicity Triterpenoids compounds such as: la, 30-dihydroxy- cycloart-24-ene-30-carboxylic acid, Methyl quadrangularate c, la, 3P-dihydroxy- cycloart-24-ene-30-carboxylic acid methyl ester, Flavonoids group : Isokaempferide,

5.4'-dihydroxy-3,7,3'-trimethoxyflavone, Pachypodol, Group Miscellaneous: Methyl

gallate, p-sitosterol glucoside, p-sitosterol4

With the sameanti-cancer results, in 2000, theteam ofToyama University(Japan)has found seven compounds as cycloartan saponin triterpene from the leaves of c

shown the effects ofcombretastatin in preventing blood flow from transferring oxygen

to cancer cells, making them in starvation of oxygen that can not be developed The

studyhas brought hope to cancer patients orpeople at risk of cancer from the common

trees10

1.2.2.5 Liver protection activity

In addition to the anti-cancer results, the team of Toyama University (Japan) alsosucceeded in isolating and determining the structure of more than 30 flavovoid

compounds in the extract of methanol of leaves and seeds such as quadrangularol B,

15

Chapter 1 Literature review

kamatakein, trihydroxy -dimetoxyflavone,methyl quadrangulateA, nor quadrangularic acid, vitexin, betulinic acid These compounds are effective against the damaging

agents in livercells (Tumor Necrosis Factor Alpha, TNF Alpha) So, the leaves of Tram

Bau are effective protect liver cells10 According to research by Yasuhiro Tezuka andcoworkers (2000), reported three new lupane triterpenes, acid 2R, 6p-dihydroxybetulinic 60-hydroxyhovenic acid andone oleanane, 6p-hydroxyarjunic acid,and other compounds isolated from methanol (MeOH) extract of c quadrangulare

seeds The structure of thesecompoundswas clarified on spectral analysis andthe ability

ofcancercells killing to protectthe liver, which has been tested of cell death caused by

D-GalN/TNF-Rin cultured rat livercells

The methanol extract of the leaves showed significant liver protection effects on

D-galactosamine (D-GalN)/lipopolysaccharide (LPS) due to liverdamage in mice and

on D-GalN/Tumor (TNF-a) causescell death in cultured rat liver cells Thecompounds

isolated from methanol extract showed liver protective effects on cell death caused byD-GalN/TNF-a incultured rat liver cells Ọuadrangularol B,methyl quadrangularateI,

kamatakenin, 5,7,4'-trihydroxy-3, 3'-dimethoxyflavone, 5.4'-dihydroxy-3,7,3 '- trimethoxyflavone and isokaempferide showed strong inhibitory effects on TNF-a-induced cell death with IC50 values of 34.3, 33.7, 13.3, 22.4, 13.4 and 22.8 pM10

Compounds such as 4p, 14a-dimethyl-5a-ergosta-9p, 19-cyclo-24 hydroxy-4a-carboxylic acid, Methyl quadrilateral D, 24-epiquadrangularic M acid, 24-epiquadrangularic acid L, Norquadrangularic c and epimeric mixtures of Methyl

-en-3p-quadrangularate c and Methyl epiquadrangularate c exhibited weak liver protective

activity against cells death caused by O-GalN/TNF-a in cultured rat liver cells All of these compounds have been figured to have stronger liver protection effects than

Glycyrrhizin (a saponin-like compound with immunomodulatory, anti-inflammatory

capabilities, protect the liver and nerves)

In factnowaday, there have been many studies of extracting bioactivecompounds

onbacteria, fungi andcancer cells However, theanticanceractivity was not highorhad

no effect on different cancer cells (there was no difference between the control group

and the extract/substance)

Chapter 1 Literature review

On the other hand, the extract from Combretum quadrangulare proved to be

effective oncancer cells, mostof which were extract,meaning crude extractfrom apart

ofCombretum quadrangulare. At present, there are not many studies proving that theextract from Combretum quadrangulare has toxic effects on breast cancer cells In

addition, the premise study conducted at the Laboratory of Stem Cells and Cancer

(Nguyen Tat Thanh University) showed the ability to cause hepatocellular carcinomaand very strong blood ofthe extract from the plant (with the smallest IC50 on K562 is

5.2 pg/ml) Therefore, this study was conducted to screen for extract or segment with strong toxic effect on MCF-7 breast cancer cells,with the purpose of further extractingthe new compound also works good on breast cancercell

17

Chapter 2 Contents and methods

CHAPTER 2 CONTENTS AND METHODS

- Screening extracts or fractions ofCombretum quadrangulare at each extraction

phase based on toxic effect on breast cancer cells MCF-7 through cell morphology and IC50 values

- Determine antibacterial and antioxidant activity of the best cytotoxic fraction

2.3 Methods

2.3.1 Cells preparation

2.3.1.1 Thawing cells

MCF-7 breast cancercells vial was initially stored in liquidnitrogen tank at -196

°C In order to have cells source to carry out the subsequent procedures, MCF-7 cells

activation from liquid nitrogen is a must

Thawing process was performed by quickly taking the vial containing the cells

from the liquidnitrogentank into the waterbath at 37 °C andgently agitating until about80% of vialcontents were liquefied Thevial contents were transferredinto a centrifuge tube including prewarmedcomplete medium and centrifugedat 1000 rpm for5 minutes

at 4 °C The cell pellet was resuspended with the complete medium in T-75 Coming flask and incubated at37 °C and 5% CO2 The cells were tested for cell activation ability

- the cell attached to the bottom ofthe flask after24 hours of culture under the reverse

microscope (Optika) The cells were changed medium and checked microbial contaminationtwice a week