Down-regulation of BI-1 expression leads to prostate cancer cell death

In order to evaluate the function of BI-1 in human PC-3 and LNCaP prostate carcinoma cells the novel approach of gene silencing through RNAi was applied. RNA interference (RNAi) or RNA silencing is the process whereby double-stranded RNA (dsRNA or duplex RNA) induces the homology-dependent and specific degradation of cognate mRNA. As reviewed by Hutvagner and Zamore (2002), the specific knockdown of expression of several genes was studied in a wide variety of species, such as Caenorahbditis elegans, Drosophila melanogaster, Arabidopsis thaliana, Neurospora crassa and embryonic cells from Mus musculus. More recently, the use of RNAi has been extended to differentiated mammalian cells (Elbashir et al. 2001, Harborth et al.

2001).

To determine the function of BI-1 in vitro, human independent PC-3 and androgen-dependent LNCaP prostate carcinoma cells were transfected with small interfering double-stranded RNA (siRNA) oligonucleotides against the BI-1 gene leading to a specific down-regulation of BI-1 expression in both cell lines either at the RNA or the protein level. After treatment of PC-3 cells with duplex siRNA oligonucleotides against BI-1, the number of apoptotic PC-3 cells reached its maximum at 45 hours after treatment (35% to 44%) by using three independent methods (DAPI, ISEL and caspase-3 assay). In addition, the amount of PC-3

cell death was analyzed by trypan blue staining where induction of PC-3 cell death by duplex siRNA oligonucleotides against BI-1 peaked at 45 h after treatment (59% positively stained cells). The activation of caspase-3 paralleled the induction of apoptosis in duplex siRNA-transfected PC-3 cells. Furthermore, the increase in DNA fragmentation was almost comparable to that of trypan blue-positively stained cells, which suggests that the cytotoxicity induced by duplex siRNA against BI-1 is attributable to both necrotic and apoptotic death. However, it cannot be ruled out that trypan blue staining of PC-3 cells was accomplished due to secondary necrotic cells which are known to be readily formed from apoptotic cells over time. This hypothesis is supported by the fact that only apoptotic cells were observed after DAPI staining of transfected PC-3 cells. Further, the treatment of LNCaP cells with duplex siRNA oligonucleotides against BI-1 revealed a similar effect as in PC-3 cells at 5 days after treatment (39% of apoptotic cells).

In agreement with our results in human PC-3 and LNCaP prostate carcinoma cells, it has been previously demonstrated that the BI-1 protein inhibits Bax-induced apoptosis in mammalian cells and when ectopically expressed in yeast (Xu et al. 1998). Furthermore, it was shown that BI-1 contains six or seven predicted transmembrane domains and the localization of BI-1 was found to be similar to Bcl-2, exhibiting a nuclear envelope and endoplasmic reticulum (ER)-associated pattern. When overexpressed in human cells, an association of BI-1 with Bcl-2 and Bcl-XL was demonstrated by both chemical cross-linking and co-immunoprecipitation experiments (Xu et al.

1998). Furthermore, it is a well known fact that a potential connection exists between the close relative Bcl-2 and hormone-independent prostate cancer (Colombel et al. 1993, McDonnel et al.

1992). Moreover, BI-1 was isolated as one of the candidate suppressors of TRAIL, an apoptosis-inducing member of the tumor necrosis factor (Burns and El-Deiry 2001).

Among the various prostate cancer cell lines, recent studies demonstrated that PC-3 cells are more resistant to apoptosis than LNCaP cells (Marcelli et al. 2000, Wang et al. 1999, Chen et al.

1999). Li and coworkers (2001) reported that overexpression of Bcl-X_L underlies the molecular basis for resistance to staurosporine-induced apoptosis in PC-3 cells. Taken together, these previous findings and the results observed in the present study showing similar expression of the BI-1 gene in both PC-3 and LNCaP cells indicate that a higher apoptosis ratio in PC-3 cells as compared to LNCaP cells after treatment with siRNA oligonucleotides against BI-1 could be due to the overexpression of Bcl-X_L in PC-3 cells and an existing interaction between BI-1 and Bcl-X_L. Moreover, to support this hypothesis, pilot experiments were performed on human prostate

carcinoma DU-145 cells. As a consequence, we observed a similar apoptosis ratio in DU-145 cells (14.4%) as compared to LNCaP cells (17.4%) after 45 hours treatment with small interfering double-stranded RNA (siRNA) oligonucleotides against the BI-1 gene (Figure 35 A).

As shown in Figure 35 B, Bcl-XL is overexpressed in PC-3 cells as compared to LNCaP and DU-145 cells, therefore, it can be suggested that specific down-regulation of BI-1 (which expression is known to be associated with Bcl-X_L) leads to a higher apoptosis ratio in PC-3 cells as compared to LNCaP and DU-145. However, it can not be ruled out that these differences in the number of apoptotic cells are due to a slower cell proliferation of LNCaP cells as compared to PC-3 cells. This hypothesis is supported by the fact that a similar effect in cellular apoptosis in LNCaP cells was observed after 5 days of treatment with siRNA oligonucleotides against the BI-1 gene.

Figure 35. Cellular apoptosis mediated by siRNA oligonucleotides against the human BI-1 gene and previous reported Bcl-XL expression in human prostate cancer cells

A) Induction of prostate carcinoma cell death in prostate cancer cells (PC-3, DU-145 and LNCaP) determined in the present study by using siRNAs against the human BI-1 gene. Bar graphs represent the percentage of positively DAPI-stained apoptotic cells at 45 hours after treatment with siRNA oligonucleotides against the human BI-1 gene. Data, means of three independent experiments (+ SE). B) Overexpression of Bcl-XL in PC-3 cells (Li et al. 2001). Cell extracts (10 µg) were sized and subjected to Western analysis with antibodies for ß-actin and Bcl-XL. Arbitrary densitometric units of the Bcl-XL band were corrected for those of the ß-actin band. The ratio is reported at the bottom of each lane.

0 10 20 30 40 50

PC-3 DU-145 LNCaP

% Apoptotic Cells

A B

The results presented in this study and the previous results demonstrating in vitro binding of BI-1 with Bcl-XL and Bcl-2 indicate that down-regulation of BI-1 expression in PC-3 and LNCaP cells could change the balance of Bcl-XL/BI-1/Bcl-2 and Bax proteins and consequently, the cell death pathway can be activated as a Bax-induced apoptotic event. On the other hand, up-regulated BI-1 expression in prostate cancer cells (and other cancers) could lead to an imbalance of Bcl-X_L /BI-1/Bcl-2 and Bax proteins, thus, inhibiting programmed cell death. On the basis of our results, we conclude that down-regulation of BI-1 expression using the novel RNAi technique could serve as an attractive approach for the treatment of prostate cancer in the future.