GROUP 4

The anti-proliferative
effects of virgin coconut
oil (VCO) on colon cancer
cell line (HCT116)
De Jesus MJ, De la Cruz ARP, De Rivera MM, Del Mundo H, Dela Cruz A,
Demigillo J, Diciano D Jr., Dollete MC, Domingo XG, Doval-Santos CM,
Duro H, Edralin SL, Espiritu A, Esquivel A III, Esteban M, Fajardo RP

Dr. Nemencio Nicodemus

 INTRODUCTION

Cancer: Cocos nucifera has anticancer

3rd leading cause of mortality properties
Colon Cancer
Fatty acids in VCO with
Colorectal cancer:
6th most common type anticancer potential
2nd most common cause of VCO lauric, palmitic, linoleic,
cancer mortality linolenic, oleic
 Does virgin coconut oil have anti-proliferative
effects against HCT116 colon cancer cell line?

VCO Preparations Colon cancer cells

1. VCO-BSA 2. VCO-EtOH-BSA

Cytotoxic?
MTT Assay

Symptoms of cell health condition?

Brightfield microscopy

Senescence activity? Mode of cell death?

Flow Cytometry
β-galactosidase (Trypan blue)
assay - Annexin V-FITC Apoptotic?
- Propidium Iodide
Necrotic?
VCO Preparations

VCO treatment Negative Ctrl Positive Ctrl

Treatment 1 VCO-BSA BSA Only Doxorubicin
Treatment 2 VCO Preparations
VCO-EtOH-BSA EtOH-BSA Colon cancer
Doxurubicin
(2, 4, 6, 8, 10%) (2, 4, 6, 8, 10-) cells
1. VCO-BSA 2. VCO-EtOH-BSA
(1, 2, 2.9, 3.8, 4.7%

MTT Assay effective ethanol)

MTT Assay

Brightfield microscopy IC50: 6.61%

Flow Cytometry
β-galactosidase - Annexin V-FITC
assay - Propidium Iodide
MTT Assay

 Cocos nucifera has anticancer properties

 Fatty acids component of VCO has cytotoxic
effects:
Lauric
Palmitic
Linoleic
Linolenic
Oleic
Koschek et al., 2007; Salerno and Smith, 1991; Palombo et al., 2002;
Yasui et al., 2005; Menendez et al., 2005; Llor et al., 2003
 Brightfield microscopy
senescent
VCO treatmentcells: Negative Ctrl Positive Ctrl

Tx 1  large, flat cells with increased

VCO-BSA BSA Only granularityDoxorubicin
 increase size and number of lysozyme cell
VCO Preparations Colon cancer
enlargement and vacuolization cells
VCO-BSA
1. secondary vacuoles
2. VCO-EtOH-BSA
Robbins et al., 1970; Kurz et al., 2000; Gerland et al., 2003
MTT Assay
viable
Fig.6. (A) HCT116 cells treated with VCO-BSA
dead
Treatment (100x) (B) HCT116 cells treated with VCO-EtOH-BSA (6.61%)
Fig.2. HCT116 cells treated with BSA only
(negative control) (100x)
Fig.4. HCT116cells treates with Doxorubicin
(positive control) (100x)
VCO-BSA (200x)

Tx 2 VCO-EtOH-BSA
(8, 10-EtOH-BSA)
(2, 4, 6, 8, 10-) EtOH-BSA Doxurubicin
(2, 4, 6, 8, 10%) (1, 2, 2.9, 3.8, 4.7%
Brightfield microscopy effective ethanol)

 Ethanol is cytotoxic at very

high concentration
 lost of polyhedral shape orCytometry
Flow straight edges
β-galactosidase
Castilla et-al.,
Annexin
2004;V-FITC
Institute of Biology
assay  cell shrinkage and fragmentation
- Propidium Iodide Fig.4. HCT116cells treates with Doxorubicin

Roberts, 2000
(positive control) (100x)

Fig.3. HCT116 cells treated with EtOH-BSA negative control, higher

Fig.3. (A) HCT116 cells treated with EtOH-BSA,
Fig.5. HCT116 cells treated with VCO-
Fig.6.) HCT116
Fig.5.
concentration cells
HCT116
(8%);
EtOH-BSA (100x) treated
dead cells withtreated
cells VCO-BSA
with(200x)
VCO-EtOH-BSA (100x)
negative control (alive cells) (B) HCT116 cells
treated with EtOH-BSA, negative control, higher
concentration(dead cells)
 β-galactosidase
assay

VCO Preparations Colon cancer

cells
Trial 1
1. VCO-BSA 2. VCO-EtOH-BSA Trial 2

MTT Assay
viable dead VCO-EtOH-BSA (6.61%)
(8, 10-EtOH-BSA)
A B
Brightfield microscopy

Symptoms of Symptoms of
senescence apoptosis
C D Flow Cytometry
β-galactosidase - Annexin V-FITC
A assay
A. VCO-BSA pre-treatment B. VCO-BSA post-treatment
B VCO-BSA post-treatment
- Propidium Iodide
C C. BSA only pre-treatment D. BSA only post-treatmentD

A. VCO-BSA pre-treatment B. VCO-BSA post-treatment

C. BSA only pre-treatment D. BSA only post-treatment
VCO-BSA
VCO-BSApost-treatment
post-treatment
 β-galactosidase
assay

 chemotherapeutic agents  morphological

(vacuolization) and enzymatic changes (β-
galactosidase expression)
 possible mechanisms:
 activation of p53 and Retinoblastoma (Rb) protein
 expression of regulators: p16INK4a, p21, and
p14ARF
 inhibition of Tbx2 gene
Roninson, 2003; Sharpless and DePinho, 2004; Vance et al., 2005;
Abrahams et al., 2007
 Flow Cytometry
(Trypan Blue)
- Annexin V-FITC
- Propidium Iodide

Actions
Flow of some
cytometry fatty
VCO Preparations
Trypan blue exclusion test acids of VCO: Colon cancer
cells
 upregulate
1. VCO-BSAGADD45, p53 and PPARγ mRNA
2. VCO-EtOH-BSA

Timeand Netprotein
percentage (%)
MTT Assay
Sample viable Time (hr) Percent dead non-viable cells
VCO-EtOH-BSA (6.61%) (%)
 promote
(hr) AnnexinDNAV-FITCfragmentation
(Apoptosis) Propidium Iodide
(8, 10-EtOH-BSA) (Necrosis)
 upregulate p27Kip1
Brightfield
8% VCO-EtOH-
microscopy
8-EtOH-
VCO-EtOH-BSA EtOH-BSA BSA VCO-EtOH- EtOH-BSA
BSA
 induceSymptomsapoptosis
of Symptoms of
BSA
 1 senescence
induce 24 differentiation
cellular 13.33
apoptosis 26.67
24 61.40 15.66 4.47 Flow Cytometry 5.83
48 260.90 48 53.33 30.77
β-galactosidase - Annexin V-FITC
assay 35.88 13.7 12.21
- Propidium Iodide
3Salerno
72 Uncertain and
53.42lack 72 38.41Palombo
ofSmith, 1991;
50.00 et0.00
et al., 2002; Yasui14.20
7.06 al., 2005;
Menendez et al., 2005; Llor et al., 2003; Kemp, et al., 2003
senescence-induction
ability
 Does virgin coconut oil have anti-proliferative
effects against HCT116 colon cancer cell line?

VCO Preparations Colon cancer cells

1. VCO-BSA 2. VCO-EtOH-BSA

MTT Assay
viable dead VCO-EtOH-BSA (6.61%)
(8, 10-EtOH-BSA)

Brightfield microscopy

Symptoms of Symptoms of
senescence apoptosis
Flow Cytometry
β-galactosidase (Trypan Blue)
assay - Annexin V-FITC
- Propidium Iodide
Uncertain lack of Apoptosis : VCO-EtOH-BSA > (8, 10-EtOH-BSA)
senescence-induction Necrosis: (8, 10-EtOH-BSA) > VCO-EtOH-BSA
ability
 Recommendations
 fatty acid as positive control in cytotoxicity assay
 improve miscibility of VCO preparations
 verify probability of ethanol as extraneous
variable
 research on other cell lines, normal cells
 in vivo research
 carry out β-galactosidase assay in institutions
more familiar with testing senescence
 Recommendations
 use positive control in β-gal assay
 more sensitive assays in testing senescence
 increase number of replicates in flow cytometry
 statistical treatment
 more sensitive stains
 research probable pathways and players for cell
death