50
Western blot analysis of Bcl-2 and p53 proteins exposed to pomace extract (10, 100 and 1000 ôg/mL)
was resolved on 12% SDS-PAGE. Protein levels were significant increased in a dose- and time-dependent
manner. However, there were significant down-regulated the expression of Bcl-2 (26 kDa). These results indicate
that Shiraz red grape crudes may disturb the expression of apoptotic proteins (Figure 1).
Figure 1
Effect of Shiraz pomace extract and resveratrol on apoptotic proteins. MCF-7 cells were treated with 10-
1000 ôg/mL extract for 0 to 24 hours and treated with resveratrol for 24 hours. Protein lysates were
separated on 12% SDS-PAGE, electrotransferred onto a PVDF membrane. The membranes were
blocked and subjected to immunoblot with anti-Bcl-2 and p53, incubated with goat anti-mouse IgG-HRP
and developed with TMB substrate. Actin protein was used as protein amount control
The prevention of apoptosis is associated with the upregulation of Bcl-2 and the downregulation of Bax
(Shihab
et al
., 1999). Mutations in the Bcl-2 gene can contribute to cancers where normal physiological cell death
mechanisms are compromised by degradation of the anti-apoptotic influence of Bcl-2 (Hockenbery, 1995). Du and
Lou (2008) demonstrated the effects of grape seed extracts, catechin (100 ôM) and proanthocyanidin (100 ôM),
which activated Bcl-2, caspase-3 and caspase-9 in rat heart cell line (H9C2) for 24 h. They reported that grape
seed extracts significantly reduced the expression of Bcl-2 and activated the expression of Bax, caspase-3 (8.5-
fold) and caspase-9 (8.9-fold). Sato
et al
. (2001) also studied the effects of grape seed proanthocyanidine extract
(100 mg/kg/d) on cardiomyocyte apoptosis
in vivo
. The extract inhibited JNK-1 (46 kDa) which activates the
tumor suppressor p53. Surh
et al.
(1999) reported that resveratrol (100 ôM) in red wine decreased in the
expression of Bcl-2 protein in human promyelocytic leukemia cells (HL-60). This study showed that Shiraz
pomace extract had profound effects on the down-regulated anti-apoptotic Bcl-2 protein in MCF-7 cells for 0-24
h by dose- and time-dependent. The results were confirmed that Bcl-2 protein was originated within nucleus when
DNA was damaged. This expression may possibly relate to upregutated pro-apoptotic Bax protein which form the
pores on mitochondria outer membrane to release cytochrome c.
Conclusion
Effects of pomace extract from Shiraz grape on the phenolic compounds availability and apoptotic
actitivities were investigated. The ethanolic pomace extract contained total phenolic compounds of 1,743.504 ±
0.003 ôg of GAE/g. The study was expected that pomace extract might consider to be added-value by-products
and to be developed as therapeutic product in the future. Also, the detected effects of pomace extract could be the
10
100
1000
ȝ
g/mL
