Zinc Protoporphyrin Upregulates Heme Oxygenase-1 in PC-3 Cells via the Stress Response Pathway

Zinc protoporphyrin IX (ZnPP), a naturally occurring molecule formed in iron deficiency or lead poisoning, is a potent competitive inhibitor of heme oxygenase-1 (HO-1). It also regulates expression of HO-1 at the transcriptional level. However, the effect of ZnPP on HO-1 expression is controversial. It was shown to induce HO-1 expression in some cells, but suppress it in others. The objective of this study is to investigate the effect of ZnPP on HO-1 expression in prostate cancer PC-3 cells. Incubation of PC-3 cells with 10？μM ZnPP for 4？h showed only a slight induction of HO-1 mRNA and protein, but the induction was high after 16？h and was maintained through 48？h of incubation. Of all the known responsive elements in the HO-1 promoter, ZnPP activated mainly the stress response elements. Of the various protein kinase inhibitors and antioxidant tested, only Ro 31-8220 abrogated ZnPP-induced HO-1 expression, suggesting that activation of HO-1 gene by ZnPP may involve protein kinase C (PKC). The involvement of PKC α, β, δ, η, θ, and ζ isoforms was ruled out by the use of specific inhibitors. The isoform of PKC involved and participation of other transcription factors remain to be studied. 1. Introduction Heme oxygenase-1 (HO-1), also known as heat shock protein 32 (Hsp32), is an inducible enzyme that catalyzes the breakdown of heme, producing carbon monoxide, iron, and biliverdin. It is known to be a cytoprotective enzyme against oxidative stress [1]. It is often upregulated in tumor tissues, and its inhibition is considered as a means of sensitizing the tumors to anticancer drugs [2]. Although HO-1 expression is increased in malignant prostate tissues [3], its expression in prostate cancer cell line, PC-3, is low [4]. Induction of HO-1 expression by hemin in PC-3 cells resulted in decreased cell proliferation and migration [4]. Overexpression of HO-1 also led to nuclear location [5] and was associated with downregulation of matrix metalloprotease 9 (MMP9), which plays an important role in tumor cell invasion and angiogenesis [4]. The real function of HO-1 in tumor cells remains to be studied. HO-1 expression can be induced by many inducers, and many regulatory pathways have been proposed [6, 7]. A number of antioxidant response element (ARE)-like motifs are present in the promoter of HO-1 gene. Six of these sites were found as clusters at E1 (？3928？bp) and E2 (？9069？bp) regions of the human HO-1 promoter; they are termed StRE1 through StRE6 [6]. Besides these StRE sites, other response elements, such as HSE [8], SREBP binding site [9], and an intronic SP1