Since E was shown to affect the expression of Hsps
Since E2 was shown to affect the expression of Hsps, the transcript expression levels of another 11 Hsps were also evaluated by real-time PCR with their specific primers. The transcripts of 4 proteins (Hsp10, Hsp56, Hsp70a and Hsp110) were significantly up-regulated, but that of Hsp47 was significantly down-regulated by E2 in MCF-7 cells cultured without serum (Fig. 4A). In MCF-7 cells cultured with serum, the transcripts levels of Hsp56 and Hsp110 were significantly up-regulated, but those of Hsp40 and Hsp70b were significantly down-regulated (Fig. 4B).
The transcript levels of E2-regulated proteins identified in serum-free condition by proteomics were also evaluated in MCF-7 cells cultured with serum; those of 4 proteins, Hsp90α (U1), PDI related protein (U4), 14-3-3 protein (U14) and XTP3-transactivated protein A (U18), were significantly up-regulated (Fig. 5A). The transcript of aminoacylase 1 (D3) was significantly down-regulated, but those of Hsp60 (D1) and stathmin 1 (D6) were significantly up-regulated by E2 in serum condition (Fig. 5B). The expression of PR transcripts was induced 30-fold by E2 in MCF-7 cells cultured with serum (Fig. 5C).
Summarized above, the transcripts of 6 proteins (Hsp90α, Hsp56, Hsp110, PDI related protein, XTP3-transactivated protein A, and stathmin 1) were up-regulated and aminoacylase 1 transcript was down-regulated by E2 in MCF-7 cells cultured with or without serum. Next, to explore the temporal response of transcript expression regulation of these genes, MCF-7 cells were treated with E2 for different time, ranging from 4 to 48h (Table 4). In serum-free condition, the transcripts of Hsp56 and XTP3-transactivated protein A were up-regulated by E2 at the earlier time point, 4h. The transcripts of Hsp90α and Hsp110 were up-regulated at 8h and those of PDI related protein and stathmin 1 at 24h. However, aminoacylase 1 transcript was shown to be up-regulated at 8h, but down-regulated at 24h. In serum condition, 3 transcripts (Hsp90α, Hsp56 and XTP3-transactivated protein A) were up-regulated at 4h and 3 genes (Hsp110, PDI related protein and stathmin 1) at 24h. Aminoacylase 1 transcript was down-regulated at 24h. That is, the addition of serum changed the temporal regulation of transcript expression; E2-stimulated Hsp90α transcript was induced earlier, but Hsp110 was delayed in the presence of serum. The time courses of regulation of the known E2-stimulated genes, cyclin D1 and cyclin A2, and E2-down regulated gene cyclin G2 were also evaluated (Frasor et al., 2003); CHZ868 related genes were similarly regulated by E2 in MCF-7 cells cultured with or without serum.
Discussion Estrogens exert both direct and indirect proliferative effects on breast cancer cells in in vitro culture systems, but its sensitivity is very much influenced by culture conditions such as the presence of serum. In order to understand the cellular effect of E2 alone on transcript/protein expression, we identified the differentially expressed proteins after the treatment of E2 in MCF-7 cells cultured without serum; 26 proteins including several Hsps were differentially regulated by E2. Using real-time PCR, the transcript levels of these protein and another Hsps were measured in cells cultured with or without serum; the transcripts of 6 proteins (Hsp90α, Hsp56, Hsp110, PDI related protein, XTP3-transactivated protein A, and stathmin 1) were up-regulated and that of aminoacylase 1 was down-regulated by E2. These transcripts regulated by E2 in MCF-7 cells cultured with or without serum could be highly involved in the cellular function of E2 in breast cancer cells. Here, we discussed the potential involvement or role of these molecules in breast cancer cells. In transcript and protein levels, Hsp90α was shown to be stimulated by E2 in either the presence or absence of serum. Members of the Hsp family are molecular chaperones, which are ubiquitous proteins that act to maintain proper protein folding within the cell (Hartl, 1996). Although there is significant overlap in function between members of the molecular chaperone superfamily, Hsp90 is unique in its ability to stabilize a number of signaling proteins involved in cancer progression and it interacts with a unique set of proteins, the Hsp90 client proteins. Over 100 Hsp90 client proteins depend on Hsp90 interactions for their activity and these are involved in regulating growth and survival. A list of Hsp90 client proteins can be accessed at the homepage of Dr. Didier Picard (http://www.picard.ch/). They include chaperones and relatives (such as Hsp56 and Hsp70), transcription factors (such as p53), steroid receptors (such as ER and PR), kinases and proteins involved in cellular signaling. Since many of the proteins chaperoned by Hsp90 are involved in breast cancer progression and resistance to therapy, Hsp90 has emerged as a molecular target for breast cancer therapeutics (Isaacs et al., 2003; Beliakoff and Whitesell, 2004). There are two isoforms of human Hsp90; Hsp90α and Hsp90β. In most tissues, the Hsp90α level is elevated under conditions of cellular stress, whereas Hsp90β is constitutively expressed (Csermely et al., 1998).