Atorvastatin activates heme oxygenase-1 at the stress response elements

Abstract Statins are known to inhibit growth of a number of cancer cells, but their mechanism of action is not well established. In this study, human prostate adenocarcinoma PC-3 and breast adenocarcinoma MCF-7 cell lines were used as models to investigate the mechanism of action of atorvastatin, one of the statins. Atorvastatin was found to induce apoptosis in PC-3 cells at a concentration of 1 μM, and in MCF-7 cells at 50 μM. Initial survey of possible pathway using various pathway-specific luciferase reporter assays showed that atorvastatin-activated antioxidant response element (ARE), suggesting oxidative stress pathway may play a role in atorvastatin-induced apoptosis in both cell lines. Among the antioxidant response genes, heme oxygenase-1 (HO-1) was significantly up-regulated by atorvastatin. Pre-incubation of the cells with geranylgeranyl pyrophosphate blocked atorvastatin-induced apoptosis, but not up-regulation of HO-1, suggesting that atorvastatin-induced apoptosis is dependent on GTPase activity and up-regulation of HO-1 gene is not. Six ARE-like elements (designated StRE1 [stress response element] through StRE6) are present in the HO-1 promoter. Atorvastatin was able to activate all of the elements. Because these StRE sites are present in clusters in HO-1 promoter, up-regulation of HO-1 by atorvastatin may involve multiple StRE sites. The role of HO-1 in atorvastatin-induced apoptosis in PC-3 and MCF-7 remains to be studied.


Introduction
Atorvastatin belongs to a class of drugs known as statins, and is mainly used to lower serum cholesterol level by inhibiting the conversion of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) to mevalonate by HMG-CoA reductase, a rate-limiting step in the cholesterol biosynthesis pathway. However, this also prevents the synthesis of downstream products such as farnesyl pyrophosphate and geranylgeranyl pyrophosphate (GGPP), which are needed for post-translational activation (prenylation) of small GTPases such as Ras and Rho. Failure in the prenylation of these GTPases results in their inability to interact with a wide spectrum of functionally different downstream mediators to initiate cytoplasmic signalling pathways and to regulate cell cycle progression [1]. Hence, statins have a pleiotropic effect of inhibiting growth of a number of cancer cells, but their mechanism of action is still not well established [2]. The problem lies in the diversities of lipophilicity of statins and cell types used in the studies.
In this study, we used prostate cancer PC-3 and breast cancer MCF-7 cells as models to investigate the mechanism of action of atorvastatin. We herein report that atorvastatin up-regulates heme oxygenase-1 (HO-1) in PC-3 and MCF-7 cells.

Luciferase reporter assay
Luciferase reporter assays were carried out as described in our earlier study [25].

Data analysis
Data points shown represent mean Ϯ S.E. Statistically significant differences between data points of two groups were determined by Student's t-test. By convention, a P value of Ͻ0.05 was considered statistically significant.

Results
Atorvastatin induced significant cell death in PC-3 cells at a concentration of 1 M (data not shown). However, 10 M gave the highest cell death. All subsequent experiments using PC-3 cells were done with 10 M atorvastatin. MCF-7 cells were more resistant to atorvastatin; significant cell death was observed only when atorvastatin concentration reached to 50 M (data not shown), although significant cell death at 10 M was reported [8]. To investigate the mechanism of action of atorvastatin, luciferasereporter constructs available in the laboratory were used to test the induction of gene expression by atorvastatin. When PC-3 cells were transfected with luciferase-reporter constructs containing ARE site of NQO1 promoter, potential HSE/NF-B site of p53 promoter, FOXO site of Bim promoter or E2F-1 consensus binding site and induced with atorvastatin for 48 hrs, 2-fold induction of ARE site was observed (Fig. 1). Atorvastatin had no effect on HSE/NF-B, FOXO and E2F-1 binding sites. This suggests that atorvastatin may be involved in the Keap1/Nrf2 (nuclear factor [erythroidderived 2]-like 2) signalling pathway. To investigate which of the antioxidant response genes was induced by atorvastatin, total RNA was isolated from PC-3 and MCF-7 cells treated with atorvas-tatin, and expression of NQO1, Nrf2, HO-1, glutathione peroxidase 2 (GPX2), glutathione S-transferase 1 (GSTM1) and UDP glucuronosyltransferase 1, polypeptide A1 (UGT1A1) was evaluated by RT-PCR using gene-specific primers. Only HO-1 was found significantly induced (Fig. 2). There was also a slight increase in the expression of NQO1 and GXP2 in MCF-7 cells. The failure in detecting an increase of expression of these genes in PC-3 cells may be due to their relatively high basal expression. Minor bands of lower molecular weight HO-1 and higher molecular weight of UGT were also observed in Figure 2, these may represent unspecific PCR products or alternatively spliced mRNA. Induction of HO-1 by atorvastatin in both PC-3 and MCF-7 cells was confirmed by Western blot analysis (Fig. 3).  [26,27] (Fig. 4), similar to the results reported by other investigators using cerivastatin and other cancer cell lines [28,29]. On the other hand, ZnPP did not protect the cells from atorvastatin-induced cell death (Fig. 4). For the regulation of HO-1 expression, no significant decrease of atorvastatin-induced HO-1 mRNA in the presence of GGPP was observed (Fig. 5, as compared to Fig. 2). Surprisingly, ZnPP was a stronger inducer of HO-1 than atorvastatin, and synergistic effect with atorvastatin could be seen in MCF-7 cells (Fig. 5).

. Therefore, it is likely that HO-1 is the mediator of atorvastatin-induced cell death in these cells. To investigate the role of HO-1 in atorvastatin-induced cell death, the effect of GGPP (an intermediate in the HMG-CoA reductase pathway) and ZnPP (an inhibitor of HO-1) on induction of cell death and regulation of HO-1 expression by atorvastatin was examined. When PC-3 cells were pre-incubated with GGPP, atorvastatin-induced cell death was abrogated
Many ARE-like motifs are present in the promoter of human HO-1 gene. Six of these sites were found as clusters at E1 (Ϫ3928 bp) and E2 (Ϫ9069 bp) regions of the human HO-1 promoter, they are termed StRE1 through StRE6 [21]. StRE3 and StRE4 share identical core sequence, and StRE5 and StRE6 also share the same core sequence. The ARE site of human NQO1 promoter used in the initial experiments contains the same core sequence (5Ј-TGCTGAGTCA-3Ј) as in StRE3 and StRE4. To test the strength of theses ARE-like elements, StRE1, StRE2, StRE3 and StRE5 were subcloned into pGL3 vector and tested for induction by atorvastatin in PC-3 cells. As shown in Figure 6, StRE3 showed the highest induction level by atorvastatin, although these elements had different levels of relative luciferase activities due to different copy number of the response elements present in the luciferasereporter constructs. Besides these StRE sites, other response elements, such as HSE [22], SREBP [23] and SP1 [24] sites have also been reported to be present in HO-1 promoter. Atorvastatin did not activate the HSE, SREBP and SP1 elements (Fig. 6).   [30], macrophage [31], neuronal cells [32], liver [33], cultured human dental pulp cells [34] and vascular smooth muscle cells [35]. To the best of our knowledge, induction of HO-1 by statin in prostate and mammary cancer cells has not been reported by other investigators.

HO-1 expression but did not protect cells from atorvastatininduced apoptosis, suggesting that atorvastatin-induced apoptosis may be independent of HO-1 enzymatic activity. Up-regulation of HO-1 by statin has been reported in other cells including endothelial cells
HO-1 expression can be induced by many inducers, and many regulatory pathways have been proposed [36]. Here we reported activation of StRE in the HO-1 promoter by atorvastatin. However, the transcription factor(s) involved are not known at the present moment. Nrf2 has been shown to play an important role in the upregulation of HO-1 in the liver by simvastatin [33] and vascular smooth muscle cells by fluvastatin [37]. Interaction of Nrf2 and Bach1, two proteins of opposing action, with the StRE of human HO-1 promoter has also been reported [38]. However, the role of Bach1 in the activation of HO-1 by statin is presently unknown. Apart from the activation of StRE sites, statin has been shown to up-regulate HO-1 in macrophages via extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase G pathways [31]. It was recently reported that HO-1 was up-regulated through mRNA stabilization [39].
The role of HO-1 in cancer is controversial. On one hand, HO-1 catalyses the breakdown of heme, producing carbon monoxide, iron and biliverdin. Biliverdin is subsequently converted to bilirubin by biliverdin reductase. Carbon monoxide, biliverdin and bilirubin are antioxidants. Therefore, HO-1 is known to be a cytoprotective enzyme against oxidative stress [40]. HO-1 can be expressed at high levels in some tumour cells, and down-regulation of HO-1 by HO-1-shRNA or inhibition of the enzyme by specific inhibitor  has been shown to inhibit proliferation of some hormone-refractory prostate cancer cells [41]. On the other hand, anti-proliferative and pro-apoptotic functions of HO-1 have been reported in prostate cancer [26], breast cancer [27] and oral cancer [42], although the mechanism of action is not known. HO-1 is an integral membrane protein of the smooth endoplasmic reticulum (sER). It is anchored on the sER via a transmembrane domain at the C-terminus with most part of the protein on the cytosolic side and a short C-terminus inside the lumen of sER. However, its nuclear localization has been demonstrated by immunocytochemical and Western blot analyses [43,44]. This suggests that HO-1 may play a role in transcriptional activation of other genes. It was shown that removal of the C-terminus by proteolytic cleavage was responsible for the nuclear translocation of HO-1 [44]. HO-1 was shown to activate its own promoter and promote binding of nuclear proteins to the consensus sequences for activator protein 2 (AP-2), POU domain class 4 transcription factor, Brn3, and core-binding factor (CBF), suggesting that HO-1 plays an important role of modulating its own expression as well as the expression of other oxidant-responsive genes [44].
In summary, atorvastatin was found to induce apoptosis and up-regulation of HO-1 in PC-3 and MCF-7 cells. However, the role of HO-1 in atorvastatin-induced apoptosis in these cells remains to be studied.