Brazilein inhibits survivin protein and mRNA expression and induces apoptosis in hepatocellular carcinoma HepG2 cells

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

NI PUTU LINDA L. SITI FATIMAH KYKY HERLYANTI

Introduction
Hepatocellular carcinoma (HCC) : Evading apoptosis is a major contributor to cellular transformation, growth, and development of invasive cancer as well as drug resistance in tumors In mammalian cells, apoptosis is mainly modulated by Bcl-2 and members of the inhibition of apoptosis (IAP) family of proteins Survivin is a unique and a small (16.5kD) member of IAP family, which is associated with several subcellular compartments, and is involved in the regulation of cell death, cell cycle progression, and cell division
In this study, we report strong efficacy of brazilein in down-regulating protein and mRNA expression of survivin in human HCC HepG2 cells accompanied by caspases activation, apoptosis induction and cell growth inhibition.

Mekanisme apoptosis

Material and Methods

Extraction of Brazilien Batang 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 Culture The 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%

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 l

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

The purple-blue MTT formazan precipitate was dissolved in 100l 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).

Acrylamide Polymerization

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

Polyacrylamide Gel Electrophoresis (PAGE) Molecules are separated by size in an electric field

Smaller 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

Blotting buffer 1x Tris glycine with 20% ethanol

Electric Current

30 minutes 100V

Add the Primary Antibody

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

anti- myosin light-chain

Quickly rinse membrane in 50ml of wash buffer and discard the wash buffer

Wash

Add 50ml of wash leave for 3 minutes on the rocking platform

Add Enzyme-linked Secondary Antibody

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

Wash

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

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

Watch for Color Development

Western blot analysis

after treatment of brazilein for 48hr, cells were washed with PBS and lysed (50mM HEPES, 150mM NaCl, 1% Triton X-100, 5mM EGTA, 50mM -glycerophosphate, 20mM NaF, 1mM Na3VO4, 2mM phenyl-methyl sulfonyl fluoride, 10g/mL leupeptin and 10 g/mL aprotinin).

Cell lysates were centrifuged and the protein content was determined by Bio-Rad DC protein assay kit (Bio-Rad laboratories, Hercules, CA)

Equal amounts of protein were separated by SDSPAGE (1015%), transferred to nitrocellulose membrane and immunoblotted with antibodies as indicated.

Detection was performed using enhanced chemiluminescence (ECL) detection system

DNA ladder assay

After treatment of brazilein with different concentrations for 48hr, 1106 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

Total RNA was derived from cells with treatment of different concentrations of brazilein for 48hr. After reverse-transcription, real-time PCR (target gene and GAPDH as reference gene) was carried out in 96-well optical plates using TaqMan technology and analyzed on ABI Prism PE7700 Sequence Detection System according to standard protocols.

The primer sequences were as follows: survivin F 5GGCCCAGTGTTTCTTCTGCTT- 3; survivin R 5GCAACCGGACGAATGCTTT-3. fluorogenic probe (5FAM-AGCCAGATGACGACCCCATAGAGGAACA- 3).

Data were normalized to internal GAPDH levels and represented as relative expression (E) whereas delta Ct

PCR reaction were carried out under the following PCR conditions: 50C for 2min, and then 95C for 10min, followed by 40 cycles at 95C 15s and 60C 60s.

Results
Brazilein downregulates survivin protein and mRNA expression in HepG2 cells

Brazilein causes strong apoptotic death and growth inhibition of HepG2 cells

Conclusion
Brazilein, an important component from Caesalpinia sappan Linn. 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.

THANK YOU