gap junctional intercellular communication in cancer chemoprevention

Presented by: Naraino Majie NabiilahDate: 3rd Sept 2015

Cancer Chemoprevention by up-regulation of Gap Junctional In-

tercellular Communication

Pharmacognosy IV

• 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

RE-AGENTS

USED

DPPH Lucifer yel-low SDS Acrylamide H2O2 BHT Antibodies

METHODOLOGY

1. 1,1-diphenyl-2-picrylhydrazyl (DPPH): Scavenging activity2. Lucifer yellow: Fluorescent dye used to visualise living and fixed cells3. Sodium Dodecyl Sulfate (SDS): Used to denature proteins4. Acrylamide: Used in electrophoresis5. H2O2: Used to inhibit GJIC6. Butylated hydroxytoluene (BHT): Derivative of phenol, used for

antioxidant properties7. Antibodies: Used against MAP kinases namely Extracellular signal-

regulated protein kinases 1 and 2 (ERK1/2) that can mediate cell proliferation and apoptosis.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

METHODOLOGYPREPARATION 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


METHODOLOGYCell 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 incub-ated 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 en-hanced chemiluminescence system.

METHODOLOGY1,1-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.

RESULTS1. 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

1hGRK extract

(10mg/ml) for 30 min then H2O2 for

1h

H2O2 treatment for 1h

GOK extract (20mg/ml) for 30 min then H2O2 for

1hGRK extract

(20mg/ml) for 30 min then H2O2 for

1hD. 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

RESULTS1. 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


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 Cx43P1 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

RESULTS2. Kiwifruit extracts blocked hydrogen peroxide-induced phosphorylation of connexin 43 and extracellular signal-regulated protein kinase 1/2To 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.

RESULTS3. 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.

RESULTS4. 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.

DISCUSSIONRecently, 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.

DISCUSSIONThe 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.

DISCUSSIONUsing an automated oxygen radical absorbance capacityassay, kiwifruit was shown to have greater antioxidant activity than grapefruit, apple or pear in vitro.

Increased antioxidantactivity 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

http://www.eurekaselect.com/64292/article/gap-junctions-targets-cancer-chemoprevention-and-chemotherapy

http://www.eurekaselect.com/64292/article/gap-junctions-targets-cancer-chemoprevention-and-chemotherapy

http://cdn.intechopen.com/pdfs-wm/28028.pdf

gap junctional intercellular communication in cancer chemoprevention

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