Brazilein inhibits survivin protein and mRNA expression and induces apoptosis in

hepatocellular carcinoma HepG2 cells

X. ZHONG, B. WU, Y. J. PAN, S. ZHENG

Introduction

Mekanisme apoptosis

Extraction of BrazilienBatang kayu Caesalpinia sappan Linn. (700 g) yang tlh dikeringkan diekstraksi dengan CH2Cl2 dan aseton selama 5 hari pada suhu kamar. Ekstrak kasar diuapkan dan dimurnikan oleh QCC menggunakan heksana dan EtOAc sebagai eluen untuk memberikan brazilein murni (3,6 g).

Cell CultureThe human hepatocellular carcinoma (HCC) cell line HepG2 was obtained from the American Type Culture Collection (Manassas, VA). HCC cell lines SMMC7721 and QGY7703 were obtained from Shanghai Cell Bank (Shanghai, China)Stock solutions of brazilein in DMSO were freshly prepared for each experiment. The final concentration of DMSO in all the cultures was less than 0.2%

Material and Methods

Cell proliferation assay

5 x 103 cells were incubated in 96-well plates with different doses or absence of brazilein for 48h in a final volume of 200 μl

40 μl of MTT (5 mg/ml in PBS) was added to each well and incubated for an additional 4 h at 37 ºC

The purple-blue MTT formazan precipitate was dissolved in 100μl of DMSO.

Viability was evaluated by measuring the optical density at 570 nm on micro titer plate reader (Quant Bio-Tek Instruments, Inc.).

SDS PAGE & WESTERN BLOTTING

SDS-PAGE (PolyAcrylamide Gel Electrophoresis) SDS-PAGE, sodium dodecyl sulfate

polyacrylamide gel electrophoresis, is a technique widely used in biochemistry,

forensics, genetics and molecular biology: to separate proteins according to their

electrophoretic mobility (a function of length of polypeptide chain or molecular weight).

to separate proteins according to their size, and no other physical feature.

…SDS-PAGE SDS (sodium dodecyl sulfate) is a

detergent (soap) that can dissolve hydrophobic molecules but also has a negative charge (sulfATE) attached to it.

Fig.1Before SDS: Protein (pink line) incubated with the denaturing detergent SDS showing negative and positive charges due to the charged R-groups in the protein. The large H's represent hydrophobic domains where nonpolar R-groups have collected in an attempt to get away from the polar water that surrounds the protein. After SDS: SDS disrupt hydrophobic areas (H's) and coat proteins with many negative charges which overwhelms any positive charges the protein had due to positively charged R-groups. The resulting protein has been denatured by SDS (reduced to its primary structure-aminoacid sequence) and as a result has been linearized.

PAGE

If the proteins are denatured and put into an electric field (only), they will all move towards the positive pole at the same rate, with no separation by size.

However, if the proteins are put into an environment that will allow different sized proteins to move at different rates.

The environment is polyacrylamide. the entire process is called

polyacrylamide gel electrophoresis (PAGE).

Polyacrylamide is a useful polymer for gel electrophoresis because both the concentration and cross-linking of the gel can be controlled

Acrylamide Polymerization

Polyacrylamide Gel Electrophoresis (PAGE)Molecules are separated by size

in an electric fieldSmaller molecules migrate more rapidly through porous gel matrix

…Western Blot

Proteins are separated by gel electrophoresis, usually SDS-PAGE.

The proteins are transferred to a sheet of special blotting paper called nitrocellulose.

The proteins retain the same pattern of separation they had on the gel.

..Western Blot The blot is incubated with a generic

protein (such as milk proteins) to bind to any remaining sticky places on the nitrocellulose.

An antibody is then added to the solution which is able to bind to its specific protein.

The antibody has an enzyme (e.g. alkaline phosphatase or horseradish peroxidase) or dye attached to it which cannot be seen at this time.

..Western Blot The location of the antibody is

revealed by incubating it with a colorless substrate that the attached enzyme converts to a colored product that can be seen and photographed.

..Western Blot

Prepare to transfer proteins to a nitrocellulose membrane

• Trim gel

Mini Trans-Blot Transfer Cell

Transfer Proteins from the gel to the nitrocellulose membrane

30 minutes100V

Blotting buffer 1x Tris glycine with 20% ethanol El

ectri

c Cu

rrent

Add the Primary Antibody

anti-myosin light-chain

Wash

•Discard blocking solution•Pour 10ml of primary antibody onto the membrane and gently rock for 10 minutes•Primary antibody will bind to the myosin light-chain

•Quickly rinse membrane in 50ml of wash buffer and discard the wash buffer•Add 50ml of wash leave for 3 minutes on the rocking platform

Add Enzyme-linked Secondary Antibody

Wash

•Discard wash solution•Pour 10ml of the secondary antibody onto the membrane and gently rock for 10 minutes•Secondary antibody will bind to the primary antibody

•Quickly rinse membrane in 50ml of wash buffer and discard the wash buffer•Add 50ml of wash leave for 3 minutes on the rocking platform

Western Blot animation

Add Enzyme Substrate

Watch for Color Development

•Discard wash solution•Add 10ml of the enzyme substrate (HRP color detection reagent) onto the membrane

•Incubate for 10 minutes•The colorimetric substrate is cleaved by the enzyme conjugated (attached) to the secondary antibody

Western blot analysis

DNA ladder assayAfter treatment of brazilein with different concentrations for 48hr,

1×106 cells were collected and precipitated.

Cell pellets were washed, resuspended in cell lysis buffer (10 mM Tris-HCl [pH 7.4], 10 mM EDTA [pH 8.0], 0.5% Triton X- 100), and incubated

RNase A (0.5 mg/ml) and proteinase K (0.5 mg/ml) were added, and the pellets were incubated for 2 h.

RNase A (0.5 mg/ml) and proteinase K (0.5 mg/ml) were added, and the pellets were incubated for 2 h.

DNA was precipitated by ethanol, resuspended in distilled water and electrophoresed on a 1.5% agarose gel

The gel was stained with ethidium bromide, and the DNA was visualized using a UV transilluminater.

Quantitative real-time PCR

Results Brazilein down-regulates survivin protein and mRNA expression in HepG2 cells

Brazilein causes strong apoptotic death and growth inhibition of HepG2 cells

Brazilein, an important component from Caesalpinia sappanLinn. ethanol extract, causes a strong down-regulation of survivin protein and mRNA expression in human HCC HepG2 cells together with caspases and PARP cleavages, induction of apoptotic death and inhibition of cell growth.

Conclusion

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