1

Targeting Wnt signaling pathway in colorectal

cancer stem cells

Research internship

October 2013-February 2014

Student details Kirsten Marije Jansma s1968785 +31 646213199 k.m.jansma@student.rug.nl Department details/ External supervisor Assoc. Prof. Dr. S.C. Cheah, PhD Head of Department, Research & Innovation Faculty of Medicine & Health Sciences UCSI University, Kuala Lumpur, Malaysia cheahsc@ucsiuniversity.edu.my Faculty supervisor Prof. Dr. S. de Jong, PhD Department of Medical Oncology University Medical Center Groningen, University of Groningen

Hanzeplein 1, 9713 GZ Groningen

s.de.jong@umcg.nl

2

Abstract Colorectal cancer (CRC) is one of the most lethal types of cancer in the Western world. Even

though more effective screening methods and early detection of polyps have led to a decrease in

mortality rate, the patient’s prognosis still is poor in general. Recent studies identified the role of

cancer stem cells (CSC) in tumor initiation, growth and maintenance of colorectal cancer. These

CSC’s are less sensitive to classic cytostatic agents, therefore new targeted therapeutic

approaches have to be identified. Stem cell maintenance, cell proliferation, differentiation and

apoptosis is partly driven by canonical Wnt/β-catenin signaling pathway, thereby playing an

important role in gene transcription. Using phytochemical agents, we tried to inhibit this Wnt/β-

catenin signaling pathway in colorectal cancer stem cells. The MTT assay and iCelligence

realtime system were used to determine cytotoxicity of these agents. We found strong reduction

in cell number in HCT116 cells by all Cucurbitacins, but iCelligence was not appropriate to

measure cytotoxicity in CSC’s. By using a β-catenin activation kit, we saw that nuclear β-

translocation was reduced after treatment with Cucurbitacin B and Demethoxycurcumin.

Apoptosis, measured by Hoechst staining, was induced by Cucurbitacin B, E and

Demethoxycurcumin. A significant reduction of LGR5 protein expression was seen in samples

treated with Cucurbitacin B, E and Demethoxycurcumin. Our results suggest inhibition of Wnt

signaling pathway by Cucurbitacin B and Demethoxycurcumin. Further research is required to

determine the potential role of these agents in colorectal cancer therapy.

Samenvatting Colorectaal carcinoom is een van de dodelijkste vormen van kanker in de Westerse wereld. De

prognose is vaak slecht, ondanks betere screeningmethodes en vroege detectie van poliepen.

Recentelijk is aangetoond dat kanker stamcellen (CSC) een rol spelen in tumorigenese, groei en

voortbestaan van colorectaal carcinoom. Omdat deze CSC’s minder gevoelig zijn voor huidige

chemotherapie is het van belang om nieuwe therapieën te ontwikkelen die zich specifieker

richten op pathogenetische aspecten. ‘Wnt/β-catenin signaling pathway’ speelt een belangrijke

rol in het behoud van stamcellen, en diens proliferatie, differentiatie en apoptose. Wij

onderzochten of bepaalde fytochemische stoffen deze Wnt/β-catenin signaling remmen in

CSC’s. Eerst werd de cytotoxiciteit gemeten met behulp van MTT assay en iCelligence realtime

system. We vonden hierbij een sterke afname in aantal cellen in HCT116 cellen door de

Cucurbitacins, maar iCelligence was niet geschikt voor CSC’s. Daarna toonden wij verminderde

verplaatsing van β-catenin naar de celkern aan door Cucurbitacin B en Demethoxycurcumin.

Apoptose – gemeten met Hoechst staining - was verhoogd in de cellen die behandeld waren met

Cucurbitacin B, E en Demethoxycurcumin. Verder werd een significante vermindering van

LGR5 expressie gevonden na behandeling met Cucurbitacin B, E en Demethoxycurcumin. Onze

resultaten doen sterk vermoeden dat Cucurbitacin B en Demethoxycurcumin een remmend effect

hebben op het Wnt signaling pathway. Nader onderzoek is nodig om een eventuele rol van deze

twee middelen in de behandeling van colorectaal carcinoom vast te kunnen stellen.

3

Contents

Introduction ..................................................................................................................................... 4

Research question ........................................................................................................................... 6

Methods........................................................................................................................................... 7

Results ........................................................................................................................................... 11

Discussion ..................................................................................................................................... 18

Conclusion .................................................................................................................................... 21

References ..................................................................................................................................... 22

Appendix……………………........................................................................................................26

List of abbreviations

5-FU 5-fluorouracil

CI cell index

CRC colorectal cancer

CSC cancer stem cells

DAPI 4,6-diamino-2-phenylindole (fluorescent stain)

DMEM Dulbecco’s modified Eagle’s medium

DMSO dimethylsulfoxide

EDTA ethylenediaminetetraacetic acid

ELISA enzyme-linked immunosorbent assay

GSK-3 glycogen synthase kinase-3

IC50 concentration of drug inhibiting 50% of the cell growth

LGR5 leucine-rich repeat-containing G protein-coupled receptor 5

MTT 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltatrazolium bromide

TNM TNM Classification of Malignant Tumors; T (tumor) N (nodes) M (metastasis)

4

Introduction

Colorectal cancer (CRC) is one of the most lethal types of cancer in the United States; here,

approximately 9% of all cancer-related deaths is due to colorectal cancer1. In the Netherlands,

colorectal cancer is the third most common cancer type in men and the second most common

cancer type in women. Currently, a slight increase in incidence can be seen in Dutch population,

due to ageing and growth of the population2.

Although the mortality rate has been decreasing during the past decades, this is rather thanks to

more effective screening methods and early detection of polyps and malignancies instead of

better treatment options3. Overall five-years survival rates of colorectal cancer are on average

65%1.

The TNM-stage of the colorectal cancer is the main predictor of the prognosis. The treatment of

choice for primary CRC stage I-III is surgical resection of the tumor and its nearby located

lymph nodes and blood vessels. Adjuvant chemotherapy consisting of the FOLFOX

chemotherapy regimen after surgical resection helps to eliminate local and distant

micrometastases. Therefore it lowers the risk of disease relapse and metastasis4. The INT0035

study5 showed a 15% absolute risk reduction of recidives and a 16% absolute mortality risk

reduction in colorectal carcinoma stage III-patients when treated with 5-fluorouracil+levamisol.

Still, around 40% of the patients treated with both resection and adjuvant therapy for stage II or

III colorectal cancer will suffer from disease relapse6.

As a consequence, better therapeutic approaches have to be identified in future. Now that there

has been a lot of research in the pathogenesis of colorectal cancer, there is more understanding of

the changes in genes and proteins that lead to the development of cancer. Therefore it is

becoming less complicated to develop personalized treatment. So where chemotherapy is mainly

aiming at rapidly growing cells hence not discriminating between proliferating tumor and non-

tumor cells, targeted therapies try to attack mutated cells only. For instance, bevacizumab is a

target of Vascular endothelial growth factor (VEGF) and, when given in addition to

chemotherapy it can prolong life by 8 months7. However, even though bevacizumab is targeting

the VEGF pathway specifically, it does not mean that there are only a few side effects. A study

showed that the combination of bevacizumab with 5-fluorouracil, irinotecan and leucovorin puts

patients at higher risk for hypertension, epistaxis and wound-healing problems8.

For a long time, colorectal cancer was believed to consist of a group of tumor cells with the same

characteristics, and therefore every cell should have the same capabilities to divide and

metastasize. Recent studies identified the role of cancer stem cells (CSC) in tumor initiation,

growth and maintenance in colorectal cancer. In this theory, malignancies are the result of few

cells that have stemcell-like characteristics. Thereby, these cells can potentially be held

responsible for tumor relapse and metastasis after therapy has been given – not only in colorectal

5

cancer but in many other tumor types as well9. Just like other stem cell types, cancer stem cells

are capable of self-renewal and differentiation, and are differentiating into both the tumorigenic

and non-tumorigenic cells that are present in a tumor cell population10

. Still, researchers struggle

with the question from which cells the cancer stem cells are derived. Some assume that they are

mutated in stem cells that can be found in most tissues under normal circumstances, considering

the fact that stem cells are more prone to mutations for their capabilities of self-renewal and

extended lifespan11

.

Research has shown that cancer stem cells are less sensitive to both chemotherapeutic agents and

radiotherapy12,13

. This is also a reasonable cause of the frequent incidence of metastasis and

relapse in colorectal cancer after (adjuvant) treatment with one of these therapies. Hence,

exploring new therapeutic targets in cancer stem cells is challenging.

Stem cell maintenance, cell proliferation, differentiation and apoptosis is partly driven by

canonical Wnt signaling pathway. This pathway is a cascade of different proteins that transduce

signals from outside the cell to the nucleus. It plays a crucial role in gene transcription. In the

presence of Wnt ligands, β-catenin translocates to the nucleus, where it is essential for activating

transcription factors such as TCF4.

Excessive Wnt signaling is thought to be important in the pathogenesis of colorectal cancer. In

over 90% of all colorectal cancer cases, there is a mutation that leads to initiation of the Wnt

signaling pathway14

. As a consequence, a lot of research is now being conducted to identify

targets for the purpose of inhibiting the Wnt signaling pathway. This might be an effective

method to provoke apoptosis in cells with a constitutively activated Wnt pathway, thereby

preventing the CSC’s from excessive cell proliferation and differentiation15,16,17

. Dakeng et al.18

found a reduction of Wnt-associated proteins and intranuclear beta-catenin after inhibition of

Wnt signaling by Cucurbitacin B in breast cancer cells.

Colorectal cancer stem cells can be identified with markers such as LGR5 and CD133. Leucine-

rich repeat-containing G protein-coupled receptor 5 (LGR5) is a receptor of R-spondin family,

which promotes Wnt/β-catenin- signaling. Therefore, overexpression of LGR5 is thought to be

important in the pathogenesis of colorectal cancer, presumably by excessive Wnt/β-catenin

signaling19

. Hsu20

et al found that higher LGR5 protein levels were significantly corresponding

with higher stages in TNM classification system and with metastasis. Also, survival rates and

recurrence rates of colorectal cancer patients were higher among those with high LGR5 levels.

The response to treatment with 5-fluorouracil was higher when LGR5 expression was low, with

a 65% response rate and 37% response rate for low and high LGR5 expressing tumors

respectively. Therefore, it is assumed that in colorectal cancer the level of LGR5 expression is

correlated with the progression of the disease and the response to treatment.

6

Research question

The main question of this research is ‘Are phytochemicals effective inhibitors of the Wnt

signaling pathway in colorectal cancer stem cells?’. In this research internship we determined

whether phyotchemicals inhibit the Wnt pathway signaling and thus may be potential inhibitors

of colorectal cancer stem cell’s regulation of cell differentiation and self-renewal.

The effects of the phytochemicals were measured regarding:

- Intracellular distribution of beta-catenin with Cellomics® beta catenin activation kit

(Thermo scientific)

- Cytotoxicity assessment using MTT assay and ACEA Bioscience’s iCelligence system

- Hoechst staining was used as an indicator dye to detect apoptosis.

- LGR5 expression in the cell line was measured with colorimetric ELISA.

Hypothesis

‘LGR5-positive colorectal cancer stem cells are sensitive to phytochemicals that inhibit the Wnt-

signaling pathway’

We expected that in cell lines treated with Wnt-inhibiting compounds a smaller amount of β-

catenin is seen in the nucleus compared with control groups due to the reduced Wnt-signaling

and therefore redeuced translocation of β-catenin from cytoplasm to nucleus.

We hypothesized that the amount of cells in apoptosis is higher in cells treated with the Wnt-

inhibiting phytochemicals compared with both control groups (untreated and treated with 5-

fluorouracil). As mentioned above, it is known that cancer stem cells are less susceptible to

treatment with cytostatic agents.

Although this is not evidence-based, we supposed that there is less LGR5 expression in cell line

treated with Wnt-inhibitors. Since LGR5 is a downstream target of β-catenin, this is a plausible

assumption. Scannell et al.21

found that the degree of LGR5 expression is related to the

aggressiveness of the cancer stem cell population.

7

Methods

Cell culture

Colon cancer stem cells isolated from colorectal carcinoma cell line HCT116 were obtained

from ProMab Biotechnologies and were cultured in fresh Cancer Stem PremiumTM

medium

(ProMab) in a humidified atmosphere containing 5% CO2 at 370C. Cells were subcultured and/or

collected by using a 0.25% (v/v) trypsin-EDTA solution. Every two days, the cells were refed

with Cancer Stem Premium medium in an amount of extra 10% of the total volume

The colorectal carcinoma cell line, HCT116 (ATCC) was cultured in Dulbecco’s modified

Eagle’s medium (DMEM) containing 10% heath-inactivated fetal bovine serum (FBS), 1%

penicillin+streptomycin solution and 1% sodium pyruvate solution, also stored in a humidified

atmosphere with 5%CO2 and at 370C . A 0.25% (v/v) trypsin-EDTA solution was used to

subculture and collect the cells. The HCT116 cell line was used as control group during every

step in the protocol.

Drugs preparation

The phytochemicals used were:

1) Cucurbitacin B

2) Cucurbitacin E

3) Cucurbitacin I

4) Demethoxycurcumin

Cells also were treated with the cytostatic agent 5-fluorouracil, which is often used in the

treatment of colorectal cancer. The 5-fluorouracil served as positive control to the used

compounds. All compounds were obtained from Chromadex (Irvine – CA).

Every compound was dissolved in dimethylsulfoxide (DMSO) to a 5 mg/mL solution. Then the

compounds were aliquot and stored at -300C.

Cytotoxicity assessment by using MTT assay

The anti-proliferation abilities of the phytochemicals were assessed by using MTT (3-(4,5-

dimethylthiazol-2-yl)-2-5-diphenyltatrazolium bromide) assay. Once the confluency of the cell

line flask was 70-80%, the cell line flasks and the cancer stem cells plates were washed twice

with PBS and after trypsinization, resuspended with DMEM and Cancer Stem Premium media

for cell line and enriched stem cells, respectively. Next, the cells (at a density of 1x104 per well)

were seeded to a 96-well plate and incubated for 24 hours at 37oC with 5% CO2. Then, the cells

were treated with the phytochemical compounds, in different concentrations starting from 100

μg/mL to 3.125 μg/mL (using dilution factor 2).

After the treatment was added to the wells, the cells were cultured in the incubator for 24, 48 and

72 hours, respectively.

After the end point, cells were left incubating at 370C for 3 hours after 50 μL of MTT solution of

2 mg/mL was added to each well. Thereafter, the supernatant was discarded and 50μL of DMSO

was added to each well to dissolve the formazan product of the MTT solution. ELISA (Bio-Tek

ELx800 Absorbance Microplate Reader) was used to read the assay at the wavelength of 570nm.

8

The percentage of viability as expressed as absorbance in the presence of test compound as a

percentage of that in the vehicle control. IC50 values were calculated using GraphPad Prism 5.0

software (GraphPad software Inc) with the aid of the dose-response curve.

Cytotoxicity assessment by using realtime iCelligence System

Realtime measuring of the anti-proliferation properties of the compounds was assessed by using

the RTCA iCelligence system (ACEA Biosciences).

HCT116

Once the confluency of the culture reached 70-80%, cells were washed twice with PBS and

collected after trypsinization. 150μL of DMEM media was transferred to each well in a E-plate

L8 (ACEA), to serve as background. Then, 30000 cells in 300 μL were added to each well in the

E-plate. Overnight, the plates were kept at a 370C humidified atmosphere containing 5% CO2.

After 24 hours of incubating, 150μL of compound (dissolved in DMEM media) was transferred

to the E-plate in various concentrations. Also, for every E-plate, we kept one well as negative

control, and we transferred 150μL of a 50 μg/mL 5-Fluorouracil solution as positive control. We

chose to use Cucurbitacin B, Cucurbitacin E and Cucurbitacin I. Due to lack of time and

materials, we were not able to investigate the cytotoxicity of Demethoxycurcumin. The

cytotoxicity of the drugs were monitored every 15 minutes for 72 hours with iCelligence system.

While analyzing the outcome of the experiments, the normalization time point was set at 24

hours, right before the point where the treatment has been added.

Cancer Stem Cells

Once tumorspheres in the enriched cancer stem cell plates were clearly visible, cells were

washed twice with PBS (phosphate buffer saline) and collected after trypsinization. 150μL of

Cancer Stem Premium media was transferred to each well in a E-plate L8 (ACEA), to serve as

background. Then, 30000 cells in 300 μL were added to each well in the E-plate. Overnight, the

plates were kept at a 370C humidified atmosphere containing 5% CO2.

After 24 hours of incubating, 150μL of compound (dissolved in Cancer Stem Premium media)

was transferred to the E-plate in various concentrations. Also, for every E-plate, we kept one

well as negative control, and we transferred 150μL of a 50 μg/mL 5-Fluorouracil solution as

positive control . Here as well, Cucurbitacin B, Cucurbitacin E and Cucurbitacin I were used.

The cytotoxicity of the drugs were monitored every 15 minutes over 72 hours.

While analyzing the outcome of the experiments, the normalization time point was set at 24

hours, right before the point where the treatment has been added.

The parameter used to describe the cytotoxicity is Cell Index (CI). This is a quantitative

parameter that describes the global status of the cell, and gives information about the amount of

cells present in a well. The iCelligence system measures the impedance of the well, with Cell

Index at zero when no cells present or when no cells adhere to the well. The more cells, the

larger the CI value.

Βeta-catenin localization

Phytochemicals were assessed of the nuclear translocation of beta-catenin , by using fluorescent

beta-catenin antibody and the IN Cell Analyzer 2200 (GE Healthcare Life Sciences) .

9

When the culture reached 70-80% confluency, cells were washed twice with PBS and collected

by trypsin digestion. After, the trypsin was neutralized by DMEM or Cancer Stem Premium

media for HCT116 and enriched cancer stem cells respectively. Then, 90 μL of cell suspension

was transferred to each well of a 96-wells plate in a density of 1x104 cells per well. During 24

hours, the cells were cultured at 370C with 5%CO2. After this, the treatment was added in

various concentrations to the cells. For all compounds, a media-only well served as negative

control group. Also, GSK3 inhibitor X served as negative control, for that phosphorylation

inhibition of GSK3 is resulting in β-catenin stabilization and its subsequent translocation to the

nucleus22

,23

. The assay output parameter is the measurement of difference between the cytoplasm

intensity and nuclear intensity of β-catenin after treatment.

After 24 hours of treatment, the Cellomics Beta-Catenin Activation Kit (Thermo Scientific) was

used to detect intracellular distribution of beta-catenin and its nuclear translocation. The

performance of the experiments was according to the instructions of the manufacturer. Once the

secondary antibody was added, the further steps of the protocol were performed in a dark

environment. After the Beta-Catenin Activation Kit protocol was finished, the 96-well plates

were sealed and wrapped with aluminium foil to be kept at 4oC.

Analysis

High-content cell imaging was realized by using the IN Cell analyzer 2200 (GE Healthcare Life

Sciences). Images of each well were obtained using the IN Cell microscope through x20

objective and 341nm (DAPI), 346nm (Hoechst) and 562nm (Dylight 550) excitation filters, with

emission filters of 452nm, 497nm and 570 nm respectively.

Based on the data of the images, IN Cell 2200 Nucleus Trafficking Analysis Module (GE

Healthcare) was used to determine the quantity of beta-catenin translocation to the nucleus . This

analyzation with the Nucleus Trafficking Analysis Module was in accordance with the

manufacturer’s manual. The Collar method application inside the IN Cell 2200 Analyzer

managed to examine the fluorescence intensities of both the nucleus and cytoplasm. Thereby it

calculated the cytoplasm-to-nucleus difference.

Apoptosis

In accordance with the performance of the Cellomics Beta-catenin activation kit, Hoechst 33342

dye was added to the samples for nuclear staining. To obtain the images of the nucleus, the IN

Cell Analyzer 2200 was used. Nuclear intensity of the Hoechst 33342 was measured. Hoechst

33342 shows a brighter stain in the condensed chromatin of the apoptotic cells than the

condensed chromatin of non-apoptotic cells24

. The outcome parameter is the measurement of

nuclear Hoechst 33342 intensity.

Detection of LGR5 expression

HCT116

HCT116 cell line was subcultured into 6 new T-25 flasks. The cells were incubated until they

reached 70-80% confluency before the treatment was applied.

10

Enriched cancer stem cells

The volume of two 6-well plates containing enriched cancer stem cells with media was collected.

Then the wells were washed twice with PBS and after trypsinization, DMEM media was added

to neutralize the trypsin. The total volume was centrifuged, the supernatant was discarded and

the cell pellet was resuspended with fresh Cancer Stem Premium media. Then the suspension

was divided over two 6-well plates. The plates were kept at the incubator for two days to allow

the cancer stem cells to attach and multiply.

The amount of treatment that was added, was calculated based on the IC50 as acquired earlier

with the cytotoxicity experiments. In the case of Cucurbitacin B, E, I and Demethoxycurcumin

we used the IC50 value obtained by MTT assay. For 5-Fluorouracil, a 5 μg/mL solution was

added as a positive control.

The compounds were added to the flasks and suspended with the cells. After, the flasks were

transferred to a humidified atmosphere with 5% CO2 for a time lapse of 24 hours. After 24 hour

treatment, the treatment was stopped by collecting the cell pellet and cells were stored in cold

PBS at a 4o C environment.

Protein extraction

In order to extract membrane proteins from the samples, we used the Plasma Membrane Protein

Extraction Kit (BioVision). This kit contains buffers and reagents to efficiently extract plasma

membrane proteins specifically instead of extracting all cellular membraine proteins.

All procedures were performed according to the manufacturer’s manual. After all steps of the

protocol were completed, the plasma membrane proteins were stored at -80oC.

LGR5 ELISA protocol

Before proceeding to the LGR5 ELISA protocol, the protein that we extracted from the cell

pellets were subjected to Bradford Assay for protein quantification, and albumin was used to

generate the standard curve. After protein quantification, 20 μg/mL of each sample was used for

LGR5 assay. The Human leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5)

ELISA kit (Cusabio) was used to accurately aim the LGR5 fractions in the plasma membrane

protein samples. Also in this protocol, all steps were performed in accordance with the user’s

guide provided by the manufacturer.

Data were acquired with ELISA reader (Bio-Tek ELx800 Absorbance Microplate Reader)

reading at a wavelength of 450nm. A linearized standard curve made with standard diluents was

used to assess the quantity of LGR5 in the samples.

Statistical analysis

Each experiment was performed at least two times independently. Results were expressed as the

means value ± standard deviation (SD). Statistical analysis was performed with one-way analysis

of variance (ANOVA), with Dunnett’s Multiple Comparison Test to identify between-group

differences using GraphPad Prism software (version 5.0; GraphPad Software Inc., San Diego,

CA) . Statistical significance is expressed as ***, P<0.001 ; **, P<0.01; *, P<0.05.

Log IC50 calculations were performed using the built-in algorithms for dose-response curves with

variable slope.

11

Results

Cytotoxicity MTT

In order to assess the cytotoxicity of the phytochemical compounds on colorectal cancer stem

cells (CSC) and HCT116 cells, cultured cells were treated with different drug concentrations of

the following compounds: Cucurbitacin B, Cucurbitacin E, Cucurbitacin I, Demethoxycurcumin

and 5-Fluorouracil, the latter serving as positive control. After 24, 48 and 72 hours of treatment,

cell viability was assessed by MTT assay. LGR5 expression in HCT116 and CSC’s was first

measured, and as expected, cancer stem cells (695 pg/mL) expressed much higher levels of

LGR5 protein when compared to HCT116 cells (120 pg/mL).

In HCT116 cell line, all phytochemical compounds showed a reduction in viable cells (Figure

1A-C). In the first 24 hours of treatment,

Cucurbitacin E had the strongest cytotoxic

effect with an IC50 value of 3.18 μg/mL (Table

1). After 48 hours and 72 hours as well,

Cucurbitacin E seemed to be the most potent

drug among the four compounds in HCT116

cell line. Data suggest that the potency of

Cucurbitacin B is most pronounced when cells

are treated for a minimal time lapse of 72 hours.

Demethoxycurcumin showed a more stable

trend in IC50 value among different time lapses.

In colorectal cancer stem cells, all compounds

show a tendency to reduce the cell viability

(Figure 2A-C). But the IC50 values in the CSC

samples are obviously much higher when

compared to the IC50 values in the HCT116

cells (Table 2). Striking is the relative low IC50

value for 5-Fluorouracil , when compared to the

values of the phytochemicals. Initially,

Cucurbitacin B shows a higher potency in the

first 24 hours of treatment, but somehow

appears less potent on longer-term treatment.

The other way around, Demethoxycurcumin

becomes more powerful when given for a

longer time.

HCT116 24

hrs

48

hrs

72

hrs

5-Fluorouracil 6.402 6.35 5.771

Cucurbitacin B 17.19 9.309 8.546

Cucurbitacin E 3.180 4.597 6.984

Cucurbitacin I 20.47 18.25 12.39

Demethoxycurcumin 9.815 9.668 10.49

Table 1. The IC50 values of various compounds on cell line

HCT116. All values are expressed in μg/mL

CSC 24

hrs

48

hrs

72

hrs

5-Fluorouracil 9.26 8.62 9.74

Cucurbitacin B 6.89 33.23 35.65

Cucurbitacin E 16.59 15.92 14.38

Cucurbitacin I 32.48 73.49 84.28

Demethoxycurcumin 18.68 12.45 13.48

Table 2. The IC50 vales of the various compounds on

colorectal cancer stem cells. All values are expressed in μg/mL

12

Figure 1. Effect of the compounds on HCT116 cell line. Cell number was measured using MTT assay. A: 24 hours treatment, B: 48 hours treatment, C: 72 hours treatment.

Figure 2. The effect op the phytochemical compounds on cancer stem cells. A: after 24 hours of treatment, B: after 48 hours of treatment, C: after 72 hours of treatment

13

Cytotoxicity iCelligence

We used ACEA Bioscience’s iCelligence system to measure the cytotoxicity of Cucurbitacin B,

E and I on HCT116 cells and colorectal cancer stem cells.

In HCT116 samples (Appendix IA-IC), the control group line tends to rise after normalization

point, then reaches a plateau . This is in contrast to the samples treated with Cucurbitacin B as

well as Cucurbitacin E and I. Here, the number of cells decreased rapidly after the treatment was

added. Also, 5FU showed a strong decrease in cell number, but with an initial rise in Cell Index

after treatment was given, and a fall in CI approximately 20 hours after treatment.

In colorectal cancer stem cell samples, there is not an obvious visible difference in outcome of

Cell Index. The samples treated with the phytochemical compounds show a downfall in cell

number. Also in the control samples, a gradually decrease in cell number can be seen while we

expected the CI to increase. Therefore, we conclude that iCelligence assay is not appropriate as a

tool of measuring the impedance in colorectal cancer stem cells samples. The precise cause of

this reduction in CI is not known, though we suspect that adhesion to plates is impaired. Since

the iCelligence system is a new appliance (April 2013), no literature on this phenomenon has

been published.

Using iCelligence system, we were able to acquire the IC50, the amount of the compound that is

required in order to reach a 50% inhibition in vitro. Therefore it is a measure of the drug potency.

The IC50 we were able to obtain from iCelligence, are shown in table 3A and 3B. Striking is the

rise in IC50 value in HCT116 cells after 24 and 48 hours in Cucurbitacin E and I, even though

the graphs show stable CI immediately after drug was added to the wells. Also, even the lowest

concentration of Cucurbitacin I (3.1 μg/mL) resulted in a plateau with the same CI as these of the

higher concentrations, but with IC50 value of 4.07 μg/mL. Therefore, we suspect overestimation

of IC50 values. The IC50 values of the CSC’s are not reliable, because the iCelligence system was

not appropriate in these cells.

3A CSC 24hr 48hr 72hr

Cucurbitacin

B

3.49 23.3 23.8

Cucurbitacin

E

18.0 12.0 17.0

Cucurbitacin

I

30.6 78.5 91.6

Table 3A , B; The cytotoxicity IC50 values of the phytochemicals obtained by iCelligence system A; colorectal cancer stem

cells, B: HCT116 cell line

3B HCT116 24hr 48hr 72hr

Cucurbitacin

B

10.7 11.6 6.52

Cucurbitacin

E

3.23 5.9 75.7

Cucurbitacin

I

4.07 42.3 42.9

14

B-catenin translocation

We used the Cellomics Beta-Catenin Activation Kit to assess the level of nuclear translocation of

β-catenin in both colorectal cancer stem cells and HCT116 cells. With IN Cell Analyzer 2200 we

visualized the translocation of β-catenin and were able to quantify the nuclear intensity as well.

In HCT116 cells, we noticed a significant reduction in nuclear translocation of β-catenin in the

samples treated with Cucurbitacin B (p<0.001), Cucurbitacin E (p<0.01) , Cucurbitacin I

(p<0.05) and Demethoxycurcumin (p<0.001) (Figure 3 ). Positive control GSK3 inhibitor X

showed high nuclear translocation rates.

In colorectal cancer stem cells, Cucurbitacin B (p<0.001), Cucurbitacin E (p<0.01) and

Demethoxycurcumin treatment resulted in a significant decrease in nuclear translocation of β-

catenin (Figure 4). Even though Cucurbitacin I showed a reduction in translocation to the

nucleus, this was not statistically significant. This suggests a correlation between less effective

inhibition of growth by Cucurbitacin I and therefore less reduction in β-catenin nuclear

localization. The samples treated with GSK-3 inhibitor X showed high level of nuclear β-catenin

expression, consistent with the theory that GSK-3 inhibitor X induces the translocation of the β-

catenin to the nucleus.

Figure 3. β-catenin localization in HCT116 cells. Positive index means higher nuclear β-catenin localization while negative index means higher cytoplasmic localization. In all samples, drug concentrations were 50 μg/mL.

15

Figure 4. β-catenin localization in colorectal CSC. Positive index means higher nuclear β-catenin localization while negative index means higher cytoplasmic localization. In all samples, drug concentrations were 50 μg/mL.

Apoptosis

We measured apoptosis rate using Hoechst 33342 dye, which stains the condensed chromatins of

apoptotic cells brighter than the chromatins of non-apoptotic cells. Therefore, the more apoptotic

cells, the higher the intensity of Hoechst 33342 in the nucleus.

In HCT116 cells, apoptotic rates were significantly higher in all treated samples, with

Cucurbitacin B and I with p<0.001 and Cucurbitacin E and Demethoxycurcumin with p<0.01

(Figure 5 ). The control sample treated with 5-Fluorouracil showed a significant rise in apoptosis

as well (p<0.001), this is in line with literature that shows evidence for inducing apoptosis in

cancer cells by 5-FU25

.

In colorectal CSC samples, statistically significant increase in apoptotic rate was seen after

treatment with Cucurbitacin B (p<0.01), Cucurbitacin E (p<0.05) and Demethoxycurcumin

(p<0.05) (Figure 6). Here as well, 5-FU significantly induced apoptosis (p<0.001), consistent

with published literature26

. Cucurbitacin I did not significantly induce apoptosis in CSC’s, nor

did it give significant reduction in β-catenin nucleaur translocation as stated earlier. This

supports the relation between reduction in β-catenin translocation and apoptosis, and survival in

both CSC’s and HCT116 cells27

.

16

Figure 5. Nucleus intensity in HCT116 cells. Higher nucleus intensity suggest higher apoptosis rate. In all samples, drug concentrations were 50 μg/mL.

Figure 6. Nucleus intensity in colorectal CSC. Higher nucleus intensity suggest higher apoptosis rate. In all samples, drug concentrations were 50 μg/mL.

17

LGR5 expression

Next, we analyzed the expression of LGR5 protein by extracting proteins with the Plasma

Membrane Protein Extraction Kit (BioVision), after which the LGR5 ELISA Kit (Cusabio) was

used to measure the quantity of LGR5 proteins. Thereby, we tried to correlate the β-catenin

localization with LGR5 expression.

In colorectal stem cells, treatment with Cucurbitacin B, Cucurbitacin E, Demethoxycurcumin all

showed significant reduction (p<0.001) in LGR5 expression(Figure 7), when compared to non-

treated cancer stem cells. The sample treated with Cucurbitacin B showed the lowest expression

of LGR5 proteins. Also, the positive control 5-fluorouracil gave a significant (p<0.001) decrease

in LGR5 expression.

In HCT116 samples, not a significant difference in LGR5 protein was detected, in line with

other researches that have been performed previously. In HCT116 very low endogenous LGR5

are expressed, making it almost impossible to detect the values28,29

. Main cause for the low

LGR5 fraction in the cell line is the silencing of LGR5 by DNA methylation occurring in

HCT116 cells30

.

Figure 7. LGR5 Protein expression in Colon cancer stem cells and cell line HCT116

18

Discussion

The phytochemicals Cucurbitacins are natural compounds acquired from the cucurbitaceae

plants. Officially they are considered to be in the steroid classification and they consist of a

group of triterpoid substances renowned for their toxic effect and wide range in pharmacologic

effects. There are 12 different Cucurbitacins identified, among which Cucurbitacin B is the best

studied compound. In this study, we assessed the effects of Cucurbitacin B, E and I on Wnt

signaling pathway. We also used Demethoxycurcumin, a derivate of Cucurmin, which is a

yellow substance in the Curcuma Longa spice. Various studies identified the anti-tumor effects

of Cucurmin in a variety of solid tumors. Sandur et al.31

found that Demethoxycurcumin and

other natural derivatives of Curcumin inhibit the proliferation of various tumor types to the same

extent as Curcumin does.

Initially, we performed cytotoxicity experiments with two different mechanisms: MTT assay

and iCelligence system. We found differences in IC50 values for all compounds between MTT

assay and iCelligence experiments. This may be due to the different mechanisms of the

procedures. The mechanism of the MTT assay is dependent on NAD(P)H enzymes in

metabolically active cells that convert the MTT dye to formazan. The formazan turns the

substance purple, and ELISA microplate reader is able to measure the optical density of this

fluid. On the other hand, iCelligence system relies on the attachment of cancer cells on the layer

of the plate. The more cells, the higher the impedance. More important is the fact that the

iCelligence system appeared to be an appropriate tool to measure impedance in our colorectal

cancer stem cells, resulting in reduction in cell number even in untreated samples. Furthermore,

IC50 values in HCT116 cells didn’t seem to be correct as discussed in the results section. All in

all, this explains differences in IC50 values between the both experiments. Another conclusion

that can be drawn from the IC50 values, is that in general the IC50 is higher in the CSC sample

than the values of the same compound in the HCT116 cell line sample. This might be due to

efflux pumps – for example those from the ATP-binding cassette (ABC) family - that extrude

toxic compounds from cells, however some phytochemicals are believed to inhibit these efflux

pumps and therefore reduce resistance32

.

In our hypothesis, we stated that due to impaired translocation, we expect a smaller amount of β-

catenin in the nucleus in cells treated with possible Wnt-inhibiting agents, when compared to

control groups treated with GSK-3 inhibitor. This statement is confirmed by the data in our

study. In colorectal CSC, we found a significant reduction in β-catenin translocation to the

nucleus by all tested compounds, except for Cucurbitacin I. These results are supported by

recent literature showing that in three different human breast cancer cell lines ,Cucurbitacin B is

able to inhibit translocation of β-catenin to the nucleus. Ring et al.33

found a reduction in beta-

catenin-mediated transcription by the Wnt inhibitor VS-507 in human breast cancer stem cells,

19

leading to a reduction of growth and metastasis. The precise mechanism of degrading the Wnt/β-

catenin signaling pathway by Cucurbitacin B has yet to be clarified18

.

In connection with the hypothesis on β-catenin nuclear translocation, we expected higher

apoptosis levels in these samples treated with the phytochemicals. Indeed, we found significant

rise in apoptotic rate through Cucurbitacin B, Cucurbitacin E and Demethoxycurcumin. This can

be related to the decreased β-catenin translocation. In agreement, Cucurbitacin I did not show

significant higher apoptosis rate, consistent with lack of significant reduction in β-catenin

translocation. So far, there is no literature suggesting that Cucurbitacin I can induce apoptosis by

inhibiting Wnt signaling pathway.

Even though this hypothesis is not evidence based, our hypothesis was that LGR5 protein

expression will be lower in samples treated with the Wnt-inhibiting agents, due to the fact that

LGR5 is a target gene of the Wnt/β-catenin pathway. During this research, we observed

significantly decreased levels (p<0.001) of LGR5 expression in colorectal cancer stem cell

samples treated with Cucurbitacin B, E , I and Demethoxycurcumin. Since LGR5 is a target gene

of the Wnt signaling pathway, we may conclude that there is a significant inhibition of the Wnt

pathway. Furthermore, also in the CSC samples treated with 5-fluorouracil, a significant

reduction (p<0.001) in LGR5 expression was seen. This is consistent with a study where LGR5

positive colorectal cancer stem cells were cultured in the presence of 5-FU for 24 hours. Almost

all LGR5+ cancer cells transformed to a LGR5 negative state34

.

When we compare the results of the quantification of β-catenin nuclear translocation, apoptosis

and LGR5 expression in colorectal cancer stem cells, we may assume that both Cucurbitacin B

and Demethoxycurcumin inhibit Wnt signaling pathway. Cucurbitacin E also showed significant

reduction of β-catenin translocation to the nucleus, significant reduction of LGR5 protein

expression and significant increase of apoptotic rate, but the significance level is slightly lower

when compared to the effects of Cucurbitacin B and Demethoxycurcumin. It is not exactly

clarified whether the cytotoxicity of Cucurbitacin B, E and Demethoxycurcumin is more potent

than the cytotoxicity of 5-fluorouracil in colorectal cancer stem cells.

Apart from the effect of the phytochemical compounds on Wnt/β-catenin signaling pathway,

some studies suggest these agents can lead to changes in the sensitivity of tumor cells to

radiotherapy and cytostatic agents. Hsu et al.20

found that Cucurbitacin I in human non-small-

cell lung cancer CD133+ cells led to better response in chemoradiotherapy and suppression of

the stemness gene signatures of these cells. Moreover, administering of Cucurbitacin I was

inducing apoptosis35

. Same conclusions were drawn in a comparable study in which the

radiosensitivity was increased after Cucurbitacin I was given to patients with CD44+ head and

neck squamous carcinoma36

. Also, Cucurbitacin B enhances the effects of the cytostatic agent

cisplatin on laryngeal squamous cell carcinoma37

. Demethoxycurcumin can lead to higher

sensitivity in different solid tumor types, even when these cells are believed to be drug-

20

resistant38,39

. These studies suggest that Cucurbitacin B, E and I and Demethoxycurcumin could

play a role in adjuvant therapy in the treatment of colorectal cancer.

It is essential to have more research performed on the biologic effects of targeted therapy in

healthy cells as in cancer cells as well. Most of the side effects that are caused by these drugs

appear after long term use, and are therefore not reported during the early stages of drug

research40

. Often, it is difficult to correlate the side effects with the mechanism of the drug,

which makes it harder to link the side effects with the drugs. Few research on the side effects of

Wnt inhibition by the phytochemicals we used, has been done. However, in a study on

Cucurbitacin B in mice with pancreatic cancer, the main aim was to assess the toxicity of this

compound. In a concentration of up to 1 mg/kg bodyweight, no significant change was seen in

total body weight, organ weight of the liver, spleen and kidneys, no treatment-related toxicities

such as liver fibrosis was reported, and all blood parameters did not significantly differ from the

negative control group41

. However, for a safe procedure, it is indispensable that adverse effects

shall be identified before the drugs will be administered to patients.

In addition to cytotoxicity, β-catenin nuclear translocation, apoptosis and LGR5 protein

expression, more functional assessment of the phytochemicals could be useful to identify the

potency of the agents. C-Myc and Cyclin D1 are downstream targets of the Wnt/β-catenin

pathway. Published literature shows that Cucurbitacin B affects the expression of these Wnt

associated genes in breast cancer cell lines SKBR-3 and MCF742

and in pancreatic cancer cell

line Panc-143

. It is important to measure these downstream targets, for it gives some more

concrete information on the functional effects of Wnt/β-catenin inhibition. In colorectal cancer,

c-Myc stimulates the angiogenesis necessary for supplying the tumor with sufficient nutrients44

.

Cyclin D1 is important in cell cycle progression in transition from G1 to S phase and therefore it

partially drives replication. Some studies indicate that inhibition of Cyclin D1 could be a useful

approach for new anti-tumor therapies45

.

When we compare the effects of the Cucurbitacins and Demethoxycurcumin between HCT116

cells and CSC’s , the differences in apoptotic rate are low. A logical explanation for this

phenomenon is the anticancer effect of Cucurbitacins and Demethoxycurcumin on cancer cells

even without stem cell properties that includes drastic alterations of cell shape, resulting in

apoptosis46,47

. Also, there is not much difference in β-catenin localization between HCT116 cells

and CSC’s, of which the explanation remains unclear. LGR5 expression obviously is much more

reduced in colorectal cancer stem cells, which suggests that cancer stemness is reduced in CSC’s.

This should be confirmed by measuring stemness properties such as sphere formation.

21

Conclusion In this study, we assessed whether the phytochemicals Cucurbitacin B, E ,I and

Demethoxycurcumin are inhibitors of the Wnt signaling pathway in colorectal cancer stem cells.

Because of chemoresistance in cancer stem cells, new therapeutic approaches have to be

identified to improve the prognosis of patients suffering from colorectal cancer. The data we

found suggest that Cucurbitacin B and Demethoxycurcumin inhibit the Wnt signaling pathway.

Cucurbitacin E potentially is inhibiting Wnt pathway as well, though it is less potent than

Cucurbitacin B and Demethoxycurcumin. More studies should be performed to determine

whether these two Wnt-inhibiting agents have significant higher cytotoxicity in colorectal cancer

stem cells than classic cytostatic agents, but are less toxic to normal cells, in other words have a

larger therapeutic window. Furthermore, further research focused on synergetic interactions

between Cucurbitacin B or Demethoxycurcumin and 5-fluorouracil can provide some insights in

the possibility and benefits of combination drug treatment of these two agents.

22

References 1 Siegel R, Naishadham D, Jemal A. Cancer statistics. CA Cancer J Clin 2013; 63:11.

2 Lemmens VEPP, Coebergh JWW. Epidemiologie van colorectale tumoren. IKR Bulletin 2006; 30

(12):4-7.

3 Dennis J Ahnen; ‘Colorectal cancer: Epidemiology, risk factors, and protective factors’ .

http://www.uptodate.com/contents/colorectal-cancer-epidemiology-risk-factors-and-protective-factors .

(accessed on 14th May 2013)

4 Miguel A Rodriquez-Bigas cs,’Overview of the management of primary colon cancer’ .

http://www.uptodate.com/contents/overview-of-the-management-of-primary-colon-cancer. (accessed on

14th of May 2013)

5 Moertel CG, Fleming TR, Macdonald JS, Haller DG, Laurie JA, Goodman PJ, et al. Levamisole and

fluorouracil for adjuvant therapy of resected colon carcinoma. N Engl J Med. 1990 Feb 8;322(6):352-8.

6 Kjeldsen BJ, Kronborg O, Fenger C, Jørgensen OD. The pattern of recurrent colorectal cancer in a

prospective randomised study and the characteristics of diagnostic tests. Int J Colorectal Dis 1997;

12:329.

7 Kellie S Rath, ‘Safe and targeted anticancer therapy for ovarian cancer using a novel class of curcumin

analogs’. J Ovarian Res. 2013 May 11;6(1):35.

8 Saltz LB, Douillard JY, Pirotta N, et al . Irinotecan plus fluorouracil/leucovorin for metastatic colorectal

cancer: A new survival standard. The Oncologist 2001;6:81-91.

9 Kristel Kemper, Pramudita A Prasetyanti, Wim de Lau, Hans Rodermond, Hans Clevers, Jan Paul

Medema; ‘Monoclonal Antibodies Against Lgr5 Identify Human Colorectal Cancer Stem Cells’. Stem

Cells 2012; 30:2378-2386.

10 Hallett RM, Kondratyev MK, Giacomelli AO, Nixon AML, Girgis-Gabardo A, et al. Small Molecule

Antagonists of the Wnt/Beta-Catenin Signaling Pathway Target Breast Tumor-Initiating Cells in a

Her2/Neu Mouse Model of Breast Cancer. PLoS ONE 7(3): e33976.

11

Puglisi MA, et al. ‘Colon cancer stem cells: Controversies and perspectives’. World J Gastroenterol.

May 28, 2013 19(20): 2997–3006.

12 Baumann M, Krause M, Hill R Exploring the role of cancer stem cells in radioresistance. Nat Rev

Cancer 8: 545–554.

13Chabner BA, Roberts TG, Jr. Timeline: Chemotherapy and the war on cancer. Nat Rev Cancer 5: 65–

72.

14

Stein U, et al. . Intervening in b-catenin signaling by sulindac inhibits S100A4-dependent colon cancer

metastasis. Neoplasia, Feb 2011. 13(2): 131–144.

23

15 Kuhnert F, Davis CR, Wang HT, Chu P, Lee M, Yuan J, Nusse R, Kuo CJ. Essential requirement for

Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of

Dickkopf-1. Proc Natl Acad Sci. 2004; 101(1)

16 Pinto D, Gregorieff A, Begthel H, Clevers H. Canonical Wnt signals are essential for homeostasis of

the intestinal epithelium. Genes Dev. 2003;17:1709–1713.

17 Andrea Haegebarth, Hans Clevers; ‘Wnt Signaling and Lgr5 in Stem Cells’. AJP March 2009, Vol.

174 (3): 715-721.

18 Dakeng S et al. Inhibition of Wnt siganaling by cucurbitacin B in breast cancer cells: Reduction of Wnt

associated proteins and reduced translocation of galectin-3-mediated b-catenin to the nucleus. J Cell

Biochem. 2012 January, 113(1): 49-60.

19

Christopher A Scannell, ‘LGR5 is expressed by Ewing sarcoma and potentiates Wnt/b-catenin

signaling’ . Oncol., 15 April 2013. 3:81

20

Hsu HC, et al. (2013). Overexpression of Lgr5 correlates with resistance to 5-FU-based chemotherapy

in colorectal cancer. International Journal of Colorectal Disease, Nov 2013, Volume 28(11) :1535-1546

21

Christopher A Scannell, ‘LGR5 is expressed by Ewing sarcoma and potentiates Wnt/b-catenin

signaling’ . Oncol., 15 April 2013. 3:81

22 Metcalfe C, et al. Inhibition of GSK3 by Wnt signalling--two contrasting models. J Cell Sci. 2011 Nov

1;124(Pt 21):3537-44 23

ThermoScientific, Cellomics Beta-Catenin Activation Kits.

http://www.thermoscientific.fr/eThermo/CMA/PDFs/Product/productPDF_59314.PDF (accessed on 15-

04-2014)

24

Thermo Fisher Scientific , Hoechst 33342 Fluorescent Stain ,

http://www.piercenet.com/product/hoechst-33342-fluorescent-stain (accessed on 13-04-2014)

25

De Angelis PM . et al . Molecular characterizations of derivatives of HCT116 colorectal cancer cells

that are resistant to the chemotherapeutic agent 5-fluorouracil. Int J Oncology. 2004; Volume 24, Issue 5,

Pages 1279-1288.

26 Shakibaei, M, et al. Curcumin Enhances the Effect of Chemotherapy against Colorectal Cancer Cells by

Inhibition of NF-κB and Src Protein Kinase Signaling Pathways. PLoS One. 2013;8(2)

2727

Jiang G. et al. Targeting beta-Catenin signaling to induce apoptosis in human breast cancer cells by z-

Guggulsterone and Gugulipid extract of Ayurvedic medicine plant Commiphora mukul. BMC

Complementary and Alternative Medicine 2013, 13:203

28

Hsu, HC. et al. (2013) Overexpression of LGR5 correlates with resistance to 5-FU-based chemotherapy

in colorectal cancer. Int J Colorectal Dis (2013) 28: 1535-1546

29

Wu, C. et al. (2014). RSPO2-LGR5 signaling had tumor-suppressive activity in colorectal cancer.

Nature communications issue 5, Article number 3149, 30 january 2014

24

30

Manal R.A. Al-Kharusi, et al. LGR5 promotes survival in human colorectal adenoma cells and is

upregulated by PGE2: implications for targeting adenoma stem cells with NSAIDs.

2013. Carcinogenesis (2013) 2013 May;34(5):1150-7

31 Sandur SK, et al. Curcumin, demethoxycurcumin, bisdemethoxycurcumin, tetrahydrocurcumin and

turmerones differentially regulate anti-inflammatory and anti-proliferative responses through a ROS-

independent mechanism. Carcinogenesis. 2007 Aug;28(8):1765-73

32

Richichi C et al. Marker-independent Method for Isolating Slow-Dividing Cancer Stem Cells in Human

Glioblastoma. Neoplasia. Jul 2013; 15(7): 840–847.

33

Ring, JE et al. The Cancer Stem Cell-Targeting Wnt Inhibitor VS-507 Reduces Breast Cancer Growth

and Metastasis. Cancer Research: December 15, 2012; Volume 72, Issue 24, Supplement 3

34

Kobayashi S, et al. LGR5-Positive Colon Cancer Stem Cells Interconvert with Drug-Resistant LGR5-

Negative cells and are Capable of Tumor Reconstitution. Stem cells. 2012. Volume 30(12): 2631-2644.

35

Hsu, HS et al . Cucurbitacin I inhibits tumorigenic ability and enhances radiochemosensitivity in

nonsmall cell lung cancer-derived CD133-positive cells. Cancer. 2011; Volume 117(13): 2970-2985.

36

Chen, YW. Et al . Cucurbitacin I Suppressed Stem-Like Property and Enhanced Radiation-Induced

Apoptosis in Head and Neck Squamous Carcinoma–Derived CD44+ALDH1

+ Cells. Mol Cancer

Ther November 2010 9;2879

37 Liu, T. et al. Cucurbitacin B, a small molecule inhibitor of the Stat3 signaling pathway, enhances the

chemosensitivity of laryngeal squamous cell carcinoma cells to cisplatin. Eur J Pharmacol. 2010 Sep

1;641(1):15-22

38

Chauhan DP et al. Chemotherapeutic potential of curcumin for colorectal cancer. Curr Pharm Des 8:

1695–1706.

39

Shakibaei, M, et al. Curcumin Enhances the Effect of Chemotherapy against Colorectal Cancer Cells by

Inhibition of NF-κB and Src Protein Kinase Signaling Pathways. PLoS One. 2013;8(2)

40

Widakowich, C. et al. Review: Side effects of approved molecular targeted therapies in solid cancers.

The Oncologist, Dec 2007, Volume 12(12)1443-1455

41

Iwanski, GB. et al. Cucurbitacin B, a novel potentiator of gemcitabine with low toxicity in the treatment

of pancreatic cancer. Br J Pharmacol Jun 2010; 160(4): 998-1007

42

Dakeng, S. et al (2012). Inhibition of Wnt signaling by cucurbitacin B in breast cancer cells: Reduction

of Wnt associated proteins and reduced translocation of galectin-3-mediated b-catenin to the nucleus. J

Cell Biochem. 2012 January, 113(1): 49-60.

43

Iwanski, GB. et al (2010). Cucurbitacin B, a novel potentiator of gemcitabine with low toxicity in the

treatment of pancreatic cancer. Br J Pharmacol Jun 2010; 160(4): 998-1007

25

44

Chen, C et al . c-Myc enhances colon cancer cell-mediated angiogenesis through the regulation of HIF-

1α. Biochem Biophys Res Commun. 2013 Jan 11;430(2):505-11

45

Langenfeld J, et al . Posttranslational regulation of cyclin D1 by retinoic acid: a chemoprevention

mechanism. Proc Natl Acad Sci U S A 1997, 94(22):12070-12074

46

Lee DH et al. Cucurbitacin: Ancient compound shedding new light on cancer treatment.

heScientificWorldJOURNAL (2010) 10, 413–418

47

Huang TY et al. Demethoxycurcumin Retards Cell Growth and Induces Apoptosis in Human Brain

Malignant Glioma GBM 8401 Cells. Evidence-Based Complementary and Alternative Medicine

Volume 2012 (2012), Article ID 396573, 11 pages

26

Appendix

Figure IA. Cucurbitacin B in cell line HCT116

Figure IB. Cucurbitacin E in cell line HCT116

27

Figure IC. Cucurbitacin I in cell line HCT116

Figure ID. Cucurbitacin B in colorectal cancer stem cells

28

Figure IE. Cucurbitacin E in colorectal cancer stem cells

Figure IF. Cucurbitacin I in colorectal cancer stem cells