4.2 Effect of α -Syn knockdown against staurosporine insult
4.2.3 Expression of apoptotic markers in SK-MEL28
4.2.3.1 α-Syn knockdown up-regulates Bax expression level in SK-MEL28 To determine whether the knockdown of α-Syn expression in SK-MEL28 could further elicit apoptotic stress induced by staurosporine, the expression of downstream pro- and anti-apoptotic molecules of the intrinsic (mitochondrial) apoptotic pathway, including Bax, Bcl-2, Bcl-xL and cleaved caspase 9 were investigated. SK-MEL28 cells were incubated in the absence or presence of 0.50 µM staurosporine (LD50) for 24 hr before preparation of cell lysates and detection of apoptotic markers expression by Western blot analysis were carried out. Elevated protein level of Bax has been shown to promote apoptosis in response to numerous cell death-inducing stimuli (Oltvai & Korsmeyer, 1994). pLKO.1-HuAsynRNAi3-transfected SK-MEL28 had significantly greater endogenous Bax protein expression, as compared to untransfected SK- MEL28 (control), pLKO.1-TRC control-transfected SK-MEL28 and pLKO.1- scramble shRNA-transfected SK-MEL28 (Figure 4.7). After treatment with 0.50 µM staurosporine, Bax protein expression was markedly increased.
Higher Bax level could be observed in pLKO.1-HuAsynRNAi3-transfected SK-MEL28, as compared to that of untransfected SK-MEL28 (control), pLKO.1-TRC control-transfected SK-MEL28 and pLKO.1-scramble shRNA- transfected SK-MEL28. These results suggest that the endogenously high α- Syn expression in SK-MEL28 can attenuate the up-regulation of Bax after being subjected to staurosporine treatment and thereby protects the cells from apoptotic cell death.
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Figure 4.7: Expression of Bax in SK-MEL28 cell lines by (a) Western blot analysis and (b) its relative densitometry. Lane 1: Untransfected SK- MEL28; Lane 2: pLKO.1-TRC control-transfected SK-MEL28; Lane 3:
pLKO.1-scramble shRNA-transfected SK-MEL28; Lane 4: pLKO.1- HuAsynRNAi3-tranfected SK-MEL28. Cells were incubated in the absence or presence of 0.50 µM staurosporine for 24 hr and cell lysates were prepared and subjected to Western blot analysis. Error bars represent S.E.M from two independent experiments. * means p<0.05 compared with untransfected cells under the same treatment by one-way-ANOVA.
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4.2.3.2 α-Syn knockdown down-regulates Bcl-2 expression level in SK- MEL28
Bcl-2 has been shown to prevent both apoptotic and necrotic cell death induced by a variety of stimuli in several different systems (Zhang, Peng, Zhang, & Wu, 2003). As shown in Figure 4.8, pLKO.1-HuAsynRNAi3-transfected SK- MEL28 had significantly lower endogenous level of Bcl-2, as compared to untransfected SK-MEL28 (control), pLKO.1-TRC control-transfected SK- MEL28 and pLKO.1-scramble shRNA-transfected SK-MEL28. Bcl-2 protein expression in all four cell types was markedly increased after exposure to 0.50 µM staurosporine for 24 hr. This increased expression of Bcl-2 should be a compensatory response of the cells against the staurosporine insult. However, following staurosporine treatment, pLKO.1-HuAsynRNAi3-transfected SK- MEL28 was shown to have significantly lower Bcl-2 expression, compared with untransfected SK-MEL28 (control), pLKO.1-TRC control-transfected SK- MEL28 and pLKO.1-scramble shRNA-transfected SK-MEL28. These results suggest that endogenously high α-Syn expression in SK-MEL28 can lead to an increase of Bcl-2 expression and thereby protect the cells from staurosporine- induced cell death.
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Figure 4.8: Expression of Bcl-2 in SK-MEL28 cell lines by (a) Western blot analysis and (b) its relative densitometry. Lane 1: Untransfected SK- MEL28; Lane 2: pLKO.1-TRC control-transfected SK-MEL28; Lane 3:
pLKO.1-scramble shRNA-transfected SK-MEL28; Lane 4: pLKO.1- HuAsynRNAi3-tranfected SK-MEL28. Cells were incubated in the absence or presence of 0.50 µM staurosporine for 24 hr and cell lysates were prepared and subjected to Western blot analysis. Error bars represent S.E.M from two independent experiments. * means p<0.05 compared with untransfected cells under the same treatment by one-way-ANOVA.
4.2.3.3 α-Syn knockdown decreases Bcl-xL expression in SK-MEL28 Bcl-xL overexpression is responsible for the resistance of cells to staurosporine-induced apoptosis (Li et al., 2001). Among all four cell types tested, pLKO.1-HuAsynRNAi3-transfected SK-MEL28 had the lowest endogenous Bcl-xL protein expression, as compared to untransfected SK- MEL28 (control), pLKO.1-TRC control-transfected SK-MEL28 and pLKO.1-
Actin (45 kDa) Bcl-2 (28 kDa) Lane:
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scramble shRNA-transfected SK-MEL28 (Figure 4.9). After staurosporine treatment, the Bcl-xL level further decreased in the same trend. These results suggest that the endogenously high α-Syn expression in SK-MEL28 can lead to greater Bcl-xL expression and thereby provides protective effect against staurosporine-induced cell death.
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Figure 4.9: Expression of Bcl-xL in SK-MEL28 cell lines by (a) Western blot analysis and (b) its relative densitometry. Lane 1: Untransfected SK- MEL28; Lane 2: pLKO.1-TRC control-transfected SK-MEL28; Lane 3:
pLKO.1-scramble shRNA-transfected SK-MEL28; Lane 4: pLKO.1- HuAsynRNAi3-tranfected SK-MEL28. Cells were incubated in the absence or presence of 0.50 µM staurosporine for 24 hr and cell lysates were prepared and subjected to Western blot analysis. Error bars represent S.E.M from two independent experiments. * means p<0.05 compared with untransfected cells under the same treatment by one-way-ANOVA.
Actin (45 kDa) Bcl-xL (30 kDa) Lane:
Untreated Staurosporine-treated
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4.2.3.4 α-Syn knockdown increases staurosporine-induced caspase 9 cleavage and activation in SK-MEL28
Caspase 9 is an important member of the cysteine aspartic acid protease (caspase) family (Duan et al., 1996). Upon apoptotic stimulation, cytochrome c released from mitochondria associates with procaspase 9 (47 kDa)/Apaf-1.
This complex processes procaspase 9 into a large active subunit (35 kDa or 17 kDa) and a small subunit (10 kDa) by self cleavage at Asp315 (Li et al., 1997).
Cleaved caspase 9 further processes other caspase members, including caspase 3 and caspase 7 to initiate a caspase cascade, leading to programmed cell death (Slee et al., 1999). As shown in Figure 4.10, cleaved caspase 9 could only be detected in staurosporine-treated cells and pLKO.1-HuAsynRNAi3-transfected SK-MEL28 had the greatest cleaved caspase 9 level as compared to untransfected SK-MEL28 (control), pLKO.1-TRC control-transfected SK- MEL28 and pLKO.1-scramble shRNA-transfected SK-MEL28. These results suggest that endogenously high α-Syn expression in SK-MEL28 can attenuate cleavage and activation of caspase 9 and thereby prevent staurosporine-induced cell death.
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Figure 4.10: Expression of cleaved caspase 9 in SK-MEL28 cell lines by (a) Western blot analysis and (b) its relative densitometry. Lane 1:
Untransfected SK-MEL28; Lane 2: pLKO.1-TRC control-transfected SK- MEL28; Lane 3: pLKO.1-scramble shRNA-transfected SK-MEL28; Lane 4:
pLKO.1-HuAsynRNAi3-tranfected SK-MEL28. Cells were incubated in the absence or presence of 0.50 µM staurosporine for 24 hr and cell lysates were prepared and subjected to Western blot analysis. Error bars represent S.E.M from two independent experiments. * means p<0.05 compared with untransfected cells under the same treatment by one-way-ANOVA.
4.2.3.5 α-Syn knockdown leads to elevated Bax/Bcl-2 and Bax/Bcl-xL ratios in SK-MEL28
Bax/Bcl-2 and Bax/Bcl-xL ratios determine the apoptotic potential of the cells.
Elevated Bax/Bcl-2 and Bax/Bcl-xL ratios correspond with the onset of apoptosis (Chang et al., 2005; Raisova et al., 2001). As shown in Figure 4.11, pLKO.1-HuAsynRNAi3-transfected SK-MEL28 was shown to have
Actin (45kDa) Cleaved caspase 9 (35kDa)
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significantly elevated Bax/Bcl-2 and Bax/Bcl-xL ratios as compared to controls, both before and after staurosporine treatment. These results suggest that the endogenously high α-Syn expression in SK-MEL28 causes the cells to be less prone to apoptotic cell death induced by staurosporine.
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Figure 4.11: Ratios of (a) Bax/Bcl-2 and (b) Bax/Bcl-xL in SK-MEL28 were quantified and plotted. The Bcl-2 /Bax and Bcl-xL/Bax ratios in control (untreated and untransfected) cells were set as 1.00. Results represent mean ± S.E.M of two independent experiments. * means p<0.05 compared to untransfected cells under the same treatment by one-way-ANOVA.
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4.2.4 α-Syn knockdown reduces the proliferative index of SK-MEL28