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Abstract:
Epigallocatechin-3-gallate (EGCG) is a polyphenol from green tea extract is known to suppress the human pancreatic cancer in-vitro. Its anti-proliferative action mediated by caspase-3-activation, nuclear condensation and poly-ADP ribose polymerase cleavage. Pancreatic cancer cell death by EGCG is mediated by arresting growth at an initial stage of cell cycle. EGCG involves depolarisation mitochondrial membrane to allow cytochrome-c release into cytosal. EGCG increased the production of intracellular release oxygen species (ROS), along with C-Jun-N-terminal kinase activation in pancreatic carcinoma cells. In brief EGCG induces stress signals by substracting mitochondria and ROS-mediated JNK activation in MIA PaCa-2 pancreatic cancer cells.
Keywords: Epigallocatechin-3-gallate (EGCG), caspase-3-activation, nuclear condensation, poly-ADP ribose polymerase cleavage, ROS and MIA PaCa-2 pancreatic cancer cell.
Introduction:
Cancer is a disease of cells characterized by progressive, persistent, purposeless and uncontrolled proliferation of tissues. It has the ability to spread to other parts of the body from its origin. Green tea, popularly consuming drink around the world is known has anti-cancer activity through its polyphenols such as EGCG. EGCG is a broad spectrum anticancer activity against pancreatic carcinoma. Green tea has non-toxic activity and it is preferably in treating cancer in different organ mainly pancreatic cancer.
Materials And Methods
Materials:
- Human pancreatic carcinoma
- MIA PaCa-2
- PANC-1
- AsPc-1
- The above mentioned are growth in Petri plate, supplemented with 10% fetal bovine serum (FBS) and 50 µg/ml gentamicin at 37 degrees Celcius in 5% CO2 humidified atmosphere.
Methods:
- Soft agar assay
A total of ~20 000 cells was resuspended in 0.35% noble agar supplemented with 10% FBS and plated on 6-cm plates containing a solidified bottom layer (0.5% noble agar in growth medium). The plates were incubated in a humidified incubator at 37oC for 10–14 days. The plates were stained with 0.005% crystal violet. The colonies were counted by using an inverted microscope.
- Analysis of cell death
Chromatin condensation was determined by 4, 6-diamidino-2-phenylindole (DAPI) fluorescence as described previously. A total of 5×106 cells (in triplicate plates) was cultured in the growth medium in the presence or absence of 0.1–0.2 µM EGCG for 24 h. After fixing and permeabilization, the cells were mounted in a fluid containing 2 µg/ml DAPI (Vector Laboratories, Burlingame, CA). A Nikon Eclipse E600 fluorescence microscope (Huntley, IL) was used to visualize the nuclear stain.
- Poly-ADP ribose polymerase (PARP) cleavage analysis
Cells were treated with specified concentrations of EGCG for designated time periods. After treatment, the total cellular proteins were extracted as described previously. After normalization for the total protein content, the resulting lysate was subjected to SDS–PAGE and immunoblotting with monoclonal against PARP (Pharmingen, San Diego, CA). Immunodetection was accomplished by enhanced chemiluminescence method.
- Cell cycle analysis by flow cytometry
After the designated treatment period with 0.2µMEGCG, cells were sorted by a fluorescence activated cell sorter at Ireland Cancer Centers core facility as described previously. Mitochondrial membrane depolarization study by confocal microscopy MIA PaCa-2 cells was grown in a 6-well tissue culture dish. The cells were either treated with 0.2 µM EGCG or untreated for 14 h. After treatment, the medium was replaced with serum-free medium containing 10 mg/ml JC-1, a potential-dependent J-1 aggregate forming lipophilic cation (Molecular Probes, Eugene, OR). Cells were incubated at 37oC for 10 min followed by washing with PBS. Immediately, the cells were visualized by a confocal laser-scanning microscope (Leica SP2, Bannockburn, IL). The monomer and J-aggregate forms were simultaneously excited by 488-nm argon-ion laser sources. Polarized mitochondria were marked by punctate orange-red fluorescence staining.
- Determination of cytochrome c and oligomeric Bax by immunofluorescence microscopy
For cytochrome c, control and EGCG-exposed cells were fixed and permeabilized followed by immunostaining with mouse monoclonal cytochrome c antibody as primary and CY3 conjugated goat anti-mouse IgG as secondary. To detect oligomeric Bax, immunostaining was performed with mouse anti- Bax monoclonal antibody 6A7 as primary. Slides were visualized under a Leica SP2 confocal microscope.
- Immunocomplex kinase assay
Equal amount of protein from control and EGCG-treated MIA PaCa-2 cells were subjected to immunoprecipitation with anti-human c-Jun N-terminal kinase (JNK) antibody in lysis buffer containing 10 µM HEPES, pH 7.2, 142 µM KCl, 5 µM MgCl2, 1 µM EGTA, 0.2% NP-40 and protease inhibitors. After overnight incubation at 4oC, the immunocomplex was trapped in Protein A-sepharose CL4B beads (Amersham-Pharmacia Biotech) by incubating for 2 h at 4oC. After washing, the kinase reaction for JNK was performed by incubating the immunoprecipitated proteins in 25 µM HEPES, pH 7.5, 25 µM MgCl2, 25mMb-glycerophosphate, 1 µM DTT, 0.1 µM sodium orthovanadate, 10 µM ATP, 2 ml activated JNK (Calbiochem) and 10 µCi of [g-32P] ATP. Reactions were carried out at 30oC for 15 min. Kinase reactions were terminated by boiling with Lammelis sample buffer followed by SDS–PAGE and autoradiography.
- Measurement of reactive oxygen species (ROS)
Flow cytometric determination of ROS production was carried out as described previously. MIA PaCa-2 cells were collected by trypsinization and 200 µl of cell suspension (2×106 cells/ml) was mixed with 800 µl PBS. Cells were incubated with 10 µM 5-(and-6)-chloromethyl2,7-dichlorodihydrofluorescein diacetate, acetyl ester (molecular probes) for 15 min, followed by the addition of EGCG or hydrogen peroxide. The incubation was continued for 20 min at 370 C. The oxidative burst (hydrogen peroxide) was detected using a FACScan flow cytometer with excitation and emission settings of 488 and 530 nm, respectively.
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