Gap junctional intercellular communication in cancer chemoprevention

• INTRODUCTION ON GJIC
• SCIENTIFIC JOURNAL
– BACKGROUND
– METHODOLOGY
– RESULTS
– DISCUSSION
– CONCLUSION
• REFERENCES
TABLE OF CONTENT

• Gap junctions are aqueous intercellular channels which directly
link the cytoplasmic compartments of adjacent cells.
• Gap junctional channels consist of the two juxtaposed
hemichannels called connexons, each of them constituted of six
proteic subunits composed of connexin (Cx).
• These proteins are codified by multigene family with at least 21
members and their expression is tissue specific.
INTRODUCTION

• Gap junction intercellular communication (GJIC) is considered to play
a relevant role in homeostasis of multicellular organisms by regulating
processes such as cell proliferation and cell differentiation.
• GJIC is regulated by gap junction proteins connexins, and the closure
of gap junctions is particularly mediated by phosphorylation-
modulated conformational changes of connexin 43 (Cx43)
• Multiple lines of evidence indicate that GJIC is dysregulated in most
cancer cells and that its inhibition is strongly related to carcinogenesis.
• Most tumour promoters, such as pesticides, peroxisome proliferators
and dietary additives, are reported to inhibit GJIC; however, anti-
tumour drugs can reverse GJIC disruption.
INTRODUCTION

• Cancer chemoprevention involves the chronic administration of a synthetic, natural or
biological agent to reduce or delay the occurrence of malignancy.
• Many phytochemicals are reported to have chemopreventive effects and they exhibit their
action through several mechanisms.
• One mechanism to explain the cancer-preventing activities of phytochemicals include their
impact on cell signalling processes, and particular attention has been given to the stimulatory
effects exerted on GJIC.
• It has been demonstrated that phytochemicals can up-regulate the expression of the connexin
43 gene, promoting the formation of gap junctions between neighbouring cells in a tissue and
enabling more efficient exchange of signalling molecules of low molecular weight between
those cells.
• It has been proposed that this has the effect of suppressing cells that have undergone
transformation because they are surrounded by, and in communication with, normal cells that
suppress proliferation of the transformed cell or induce apoptosis.
• Indeed. it has been demonstrated that non-tumourous cells are contact-inhibited and have
functional GJIC, whilst tumour cells have dysfunctional GJIC.
INTRODUCTION

Quercetin, the active phenolic component in kiwifruit,
prevents hydrogen peroxide-induced inhibition of gap-
junction intercellular communication
D. E. Lee et al, 2010, Quercetin, the active phenolic component in kiwifruit, prevents hydrogen peroxide-induced inhibition of gap-junction intercellular
communication, British Journal of Nutrition, 104, 164–170

BACKGROUND
•GJIC is an important
mode of cell-cell
communication which
helps in maintaining
homeostasis.
•Epidemiological studies
indicate that a diet rich in
antioxidant-containing fruits
and vegetables can reduce the
risk of cancer.
•GJIC is dysregulated
in cancer cells and its
inhibition leads to
carcinogenesis.
•Has high levels of
antioxidants [Quercetin as
main antioxidant phenolic]
•Treatment of lung, liver and
GIT (Stomach) cancers in
TCM
•Provides pretection against
oxidative DNA damage
•Enhances DNA repair
•Protects against mutagenic
changes.
Inhibited sarcoma 180 in
mice by 30-40%
On a fresh-weight basis; Total phenol
content = 274 mg/100 g
0·63–1·06 mg quercetin
glycosides/l

1. Epidemiological studies have
shown that quercetin consumption
reduces the risk of developing cancer.
2. Further, the chemopreventive activity of
quercetin has been demonstrated in a
variety of laboratory animal models,
including azoxymethane-induced colonic
tumorigenesis in mice.
3. Additionally, quercetin is reported to
prevent the appearance of pre-neoplastic
lesions in rat hepatocarcinogenesis
BACKGROUND

• Particularly, H2O2 is a well-known cancer promoter that disrupts GJIC.
• H2O2 inhibits GJIC in WB-F344 rat liver epithelial cells with a 50% inhibition
(I50) value of 200mM.
• WB-F344 cells are stimulated directly by H2O2 and H2O2 promotes
proliferation and transformation of WB-F344 cells.
• H2O2-mediated interference of GJIC has been reported to particularly
correlate with the phosphorylation of Cx43 and extracellular signal-regulated
protein kinase (ERK).
• The present study was designed to investigate the effects of both the gold
kiwifruit (GOK) and green kiwifruit (GRK) cultivars, and their active phenolic
phytochemical quercetin, on the H2O2-mediated inhibition of GJIC.
BACKGROUND

REAGENTS
USED
DPPH Lucifer yellow SDS Acrylamide H2O2 BHT Antibodies
METHODOLOGY
1. 1,1-diphenyl-2-picrylhydrazyl (DPPH): Scavenging activity
2. Lucifer yellow: Fluorescent dye used to visualise living and fixed cells
3. Sodium Dodecyl Sulfate (SDS): Used to denature proteins
4. Acrylamide: Used in electrophoresis
5. H2O2: Used to inhibit GJIC
6. Butylated hydroxytoluene (BHT): Derivative of phenol, used for antioxidant
properties
7. Antibodies: Used against MAP kinases namely Extracellular signal-regulated
protein kinases 1 and 2 (ERK1/2) that can mediate cell proliferation and apoptosis.

METHODOLOGY
PREPARATION OF KIWIFRUIT EXTRACTS
Two main
kiwifruit
cultivars, GOK,
which has a
yellow colour and
non-astringent
taste, and GRK,
which has a green
colour and
astringent taste
were carefully
pared, frozen and
dried.
The freeze
dried
kiwifruits
were ground
to powder
and stored at
220C until
used.
Kiwifruit extracts
were generated
by mixing 10 g
lyophilised
kiwifruit with 100
ml of 80%
aqueous
methanol.
The kiwifruit–
methanol
mixture was
sonicated for
20 min with
continuous
N2 gas
purging.
The mixture was
filtered through
Whatman no. 2
filter paper using
a chilled Buchner
funnel and then
rinsed with 50 ml
methanol.

PREPARATION OF KIWIFRUIT EXTRACTS
The solid filter
cake was then re-
extracted by
repeating the
above steps under
the same
conditions.
The two
filtrates were
combined,
and an
additional 50
ml of 80%
aqueous
methanol was
added.
The solvent was
evaporated using
a rotary
evaporator under
reduced pressure
at 40oC.
The extract
was dissolved
in 50 ml of
100%
methanol and
made up to
the final
volume of 100
ml with
distilled
deionised
water.
The solution was
then centrifuged
for 20 min. The
final extracted
product was
stored at 24oC
until used.
METHODOLOGY

METHODOLOGY
Cell culture
• WB-F344 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10%
fetal bovine serum and penicillin–streptomycin at 37oC in a 5% CO2 humidified incubator
Bioassay of gap-junction intercellular communication
•GJIC was measured by the scrape-loading–dye-transfer technique.
•WB-F344 cells were pre-incubated with kiwifruit extract or quercetin for 30 min and then
stimulated with 100 mM-H2O2 for 1 h.
•After H2O2 treatment, cells were washed twice with phosphate buffered saline (PBS).
•Next, lucifer yellow was added to the washed cells, and three scrapes were made using a scalpel
with a surgical-steel blade under low light intensity.
•Each scrape traversed a large group of confluent cells.
•After 3 min of incubation, the cells were washed four times with PBS and then fixed with a 4%
formalin solution.
•Communicating cells showing green fluorescence were distinguishable from cells that are not
communicating under an inverted fluorescence microscope.
•The number of communicating cells was counted.

METHODOLOGY
Western blot analysis
• Western blot analysis was performed to measure the protein level
of Cx43, ERK1/2 and phosphorylated ERK1/2.
• Briefly, total cell lysates were suspended in a sample buffers,
heated at 95oC for 5 min and separated by SDS–PAGE on a 12·5%
polyacrylamide gel.
• The proteins were then transferred to a 0·45 mm polyvinylidene
fluoride transfer membrane and incubated in a blocking buffer.
• Blots were probed with primary antibodies and then horseradish
peroxidase-conjugated goat anti-mouse IgG secondary antibodies.
• For visual detection, blots were developed using an enhanced
chemiluminescence system.

METHODOLOGY
1,1-Diphenyl-2-picrylhydrazyl radical-scavenging activity assay
• The DPPH radical-scavenging activities of resveratrol and BHT were measured
using the method described by Brand-Williams et al. with minor modifications.
• DPPH was dissolved in 80% aqueous methanol.
• A 0·1 ml volume of quercetin or BHT at various concentrations was added to 2·9ml
of the DPPH radical solution.
• The mixture was then shaken vigorously and incubated in the dark at 23oC for 30
min. Absorbance at 517 nm was measured using a spectrophotometer.
Statistical analysis
• All experiments were repeated at least three times unless otherwise stated.
• Results are presented as mean values and standard deviations of triplicates.
• Comparisons between two groups were analysed using Student’s t test.
• Probability values of P<0·05 were considered statistically significant.

RESULTS
1. Kiwifruit extracts prevented hydrogen peroxide-induced inhibition of gap-junction
intercellular communication
•The effects of two kiwifruit extracts on the H2O2-mediated inhibition of GJIC in WB-F344
cells were assessed using the scrape-loading–dye-transfer technique.
•The distance that the dye travelled perpendicular to the scrape was observed under an
inverted fluorescence microscope as shown in Fig. 1(a).
Untreated control for
30min
GOK extract
(10mg/ml) for 30 min
then H2O2 for 1h
GRK extract (10mg/ml)
for 30 min then H2O2
for 1h
H2O2 treatment for 1h
GOK extract
then H2O2 for 1h
GRK extract
then H2O2 for 1h
D. E. Lee et al, 2010, Quercetin, the active phenolic component in kiwifruit, prevents hydrogen peroxide-induced inhibition of gap-junction intercellular communication, British
Journal of Nutrition, 104, 164–170

RESULTS
1. Kiwifruit extracts prevented hydrogen peroxide-induced inhibition of gap-
junction intercellular communication
•The number of communicating cells was
counted, and the number of communicating
cells in the untreated control was normalised to
100 %.
•The relative rate of GJIC for each of the
treatment conditions was calculated as the
percentage of the untreated control.
•Treatment of cells with 100mM-H2O2 for 1 h
reduced GJIC to about 35% (Fig. 1(b-ii)).
•However, pre-treatment of cells with 20 mg/ml
of either GOK or GRK extracts completely
prevented the H2O2-induced inhibition of GJIC
D. E. Lee et al, 2010, Quercetin, the active phenolic component in kiwifruit, prevents hydrogen peroxide-induced inhibition of gap-junction intercellular communication, British
Journal of Nutrition, 104, 164–170

Pre-treatment with 20 mg/ml of GOK
or GRK extracts for 30 min blocked the
H2O2-mediated hyper-phosphorylation
of Cx43, measured as a decrease in the
(P2+P3):(P0+P1) ratio.
RESULTS2. Kiwifruit extracts blocked hydrogen peroxide-induced phosphorylation of connexin
43 and extracellular signal-regulated protein kinase 1/2
•H2O2-mediated inhibition of GJIC has been reported to correlate with the phosphorylation of
Cx43 and ERK.
•To determine if kiwifruit extracts function by blocking the phosphorylation of these proteins,
Western blot analysis with antibodies specific to Cx43, phosphorylated ERK1/2 and total
ERK1/2 was performed.
P0 band represents non-phosphorylated Cx43
P1 is mono-phosphorylated.
P2 and P3 bands represent hyper-phosphorylated Cx43
Treatment with H2O2 caused a  in
P0 & P1 bands and an  in P3 & P4
indicating activation of hyper-
phosphorylation of Cx43

RESULTS
2. Kiwifruit extracts blocked hydrogen peroxide-induced phosphorylation of connexin
43 and extracellular signal-regulated protein kinase 1/2
To identify the molecular mechanism by which kiwifruit extracts prevent H2O2-mediated
inhibition of GJIC, the phosphorylation status of ERK1/2 was examined.
Treatment of WB-F344 cells with 20
mg/ml of either GOK or GRK for 30 min
had no effect on the phosphorylation of
ERK1/2.
In contrast, treatment with 100mM-
H2O2 for 1 h induced ERK1/2
phosphorylation.
However, pre-treatment of WB-F344
cells with 20 mg/ml of GOK and GRK for
30 min blocked H2O2-induced
phosphorylation of ERK1/2.

RESULTS
3. Prevention and blocking of H2O2 induced inhibition of GJIC and phosphorylation of
Cx43 and ERK1/2.
To determine if
purified quercetin
prevented H2O2
induced inhibition of
GJIC and blocked the
phosphorylation of
Cx43 and ERK1/2, the
previous experiments
were repeated with
pure quercetin.

RESULTS
4. Free radical-scavenging activity of quercetin
•Free radical-scavenging activity of quercetin was measured and its activity was compared
with BHT, a synthetic phenolic antioxidant used in foods.
•The amount of free radicals produced by DPPH alone was normalised to 100 %, and
increasing amounts of BHT or quercetin were added to assess their activities.
Both BHT and quercetin exhibited dose-dependent
free radical-scavenging activity.
Importantly, 50 or 100mM-quercetin efficiently
scavenged the free radicals produced by DPPH and had
superior scavenging activity to that of BHT.
As shown above, pre-treatment of cells with these
concentrations of quercetin suppressed the
phosphorylation of Cx43 and ERK1/2, thereby
preventing the inhibition of GJIC and suggesting a
possible link between the free radical-scavenging
activity and protection of GJIC.

DISCUSSION
Further, GOK- or GRK-
treated cells had a
reduced level of Cx43
and ERK1/2
phosphorylation upon
H2O2 treatment, and
phosphorylation of
these proteins is known
to be associated with
impaired GJIC.
WB-F344 cells that
were pre-treated
with an extract from
either the GOK or
GRK cultivar
maintained normal
GJIC after challenge
with 100mM-H2O2.
Quercetin also had
substantial free radical-
scavenging activity, which
was significantly greater than
the synthetic phenolic
antioxidant BHT.
Together, these data suggest
that the protective effects of
quercetin on GJIC may be
mediated by its antioxidant
properties.

DISCUSSION
Recently, attention
has been focused on
phytochemicals,
which are non-
nutritive components
of plant-based diets
that possess cancer-
preventive properties
These phytochemicals
act to prevent cancer by
blocking the initiation
or reversing the
promotion of
carcinogenesis
Quercetin is an antioxidant
found in substantial
quantities in kiwifruits,
suggesting that quercetin
may be the primary
phytochemical in kiwifruits
that is responsible for the
cancer-preventative
properties.

DISCUSSION
The mechanism by
which oxidative stress
inhibits GJIC involves
conformational
changes in gap
junctions due to the
phosphorylation of
Cx43, a major
component of gap-
junction channels.
Inhibition of GJIC also
involves the activation of
mitogen-activated
protein kinases (MAPK).
In particular,
phosphorylation of ERK
was reported to play a
key role in the inhibition
of GJIC in vitro
Previous studies demonstrated
that phosphorylation of Cx43
and ERK1/2 are important events
controlling H2O2-induced
inhibition of GJIC in WB-F344.
Kiwifruit extracts from both
GOK and GRK, and quercetin
alone, blocked H2O2-induced
phosphorylation of ERK1/2 and
Cx43, thereby protecting GJIC in
WB-F344 cells.

DISCUSSION
Using an automated
oxygen radical
absorbance capacity
assay, kiwifruit was
shown to have greater
antioxidant activity
than grapefruit, apple
or pear in vitro.
Increased antioxidant
activity was also found in
the plasma of human
subjects fed kiwifruit juice,
demonstrating the
bioavailability of kiwifruit
antioxidants in man.
The antioxidant activity of
kiwifruit might play a role in
the suppression of H2O2-
induced phosphorylation of
ERK1/2 and Cx43.
This is supported by the
superior free radical-
scavenging activity of
quercetin over BHT. Together,
these data suggest that the
antioxidant properties of
quercetin may be responsible
for the suppression of H2O2
induced phosphorylation of
ERK1/2 and Cx43 and
prevention of GJIC inhibition.

CONCLUSION
Kiwifruit extracts prevented
H2O2-induced inhibition of GJIC
by blocking the oxidative stress-
induced activation of the ERK1/2
and Cx43 signalling pathway.
The present results suggest that
the consumption of kiwifruit or
adding quercetin as a dietary
supplement might be an effective
means to lowering the risk of
developing cancer.

• J.E. Trosko and R.J. Ruch, Gap Junctions as Targets for Cancer Chemoprevention and Chemotherapy.
Available at: http://www.eurekaselect.com/64292/article/gap-junctions-targets-cancer-
chemoprevention-and-chemotherapy#sthash.zVHGn5oK.dpuf
• Glaucia M. Machado-Santelli and Marisa Ionta, Gap Junction Intercellular Communication and
Connexin Expression Profile in Normal Liver Cells and Hepatocarcinoma, Hepatocellular Carcinoma –
Basic Research. Available at: http://cdn.intechopen.com/pdfs-wm/28028.pdf
• Timothy J. King a, John S. Bertram, 2005, Connexins as targets for cancer chemoprevention and
chemotherapy, Biochimica et Biophysica Acta 1719 (2005) 146 – 160
• Dong Eun Lee, Bong Jik Shin, Haeng Jeon Hur, Jong Hun Kim, Jiyoung Kim, Nam Joo Kang, Dae Ok
Kim, Chang Yong Lee, Ki Won Lee and Hyong Joo Lee, Quercetin, the active phenolic component in
kiwifruit, prevents hydrogen peroxide-induced inhibition of gap-junction intercellular
communication, British Journal of Nutrition (2010), 104, 164–170
REFERENCES

Gap junctional intercellular communication in cancer chemoprevention

Gap junctional intercellular communication in cancer chemoprevention

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