ZnPP is relatively nontoxic to PC-3 cells. In fact, it induced significant cell proliferation at a concentration of 0.6–10μ
M, and only suppressed cell growth above 10μ
M (). Therefore, 10μ
M ZnPP was used for all subsequent experiments. Basal expression level of HO-1 protein in PC-3 was undetectable. ZnPP induced HO-1 protein expression in a dose-dependent manner, with the highest induction level at 10μ
M (). For a time course study, incubation of the cells with 10μ
M ZnPP for 4
h showed only a slight induction of HO-1 protein, but the induction was high after 16
h and was maintained through 48
h of incubation (). The HO-1 mRNA level as determined by RT-PCR also showed similar profile ().
Figure 1 Effect of ZnPP on cell proliferation and HO-1 expression of PC-3 cells. (a) PC-3 cells were treated with various concentrations of ZnPP for 48h, and number of live cells was estimated by MTS cell proliferation assay as described in Section 2 (more ...)
Figure 2 Induction of HO-1 by ZnPP in PC-3 for various lengths of time. PC-3 cells were treated with 10μM ZnPP for 0.5, 1, 4, 16, 24, and 48h (lanes 2, 3, 4, 5, 6, and 7, resp.). Control cells were treated with equal amount of DMSO for (more ...)
Of all the known responsive elements in the HO-1 promoter, ZnPP activated mainly the ARE-like elements (StREs). As shown in , StRE3 showed the highest (6.6-fold) induction level by ZnPP, although these elements had different basal expression levels of relative luciferase activities due to different copy number of the response elements present in the luciferase-reporter constructs. ZnPP did not activate the HSE, SREBP, and SP1 elements ().
Figure 3 Effect of ZnPP on StRE1, StRE2, StRE3, StRE5, HSE, SREBP, and SP1 elements of human HO-1 promoter in PC-3 cells. PC-3 cells were transfected with enhancer-luciferase reporter plasmid harboring one of these elements, treated with 10μM (more ...)
A number of protein kinases are known to be involved with the activation of antioxidant response element. To investigate the effect of various protein kinase inhibitors and antioxidant on the activation of StRE by ZnPP, cells transfected with StRE3-pGL3 were pretreated for 2
h with SB203580 (p38-MAPK inhibitor), LY294002 (phosphatidylinositol 3-kinase inhibitor), U0126 (MEK inhibitor), SP600125 (JNK inhibitor), IPA-3 (p21-Activated Kinase Inhibitor III), NAC (antioxidant), rottlerin (PKC-δ
inhibitor), Ro 31-8220 (pan PKC inhibitor), or Ro 32-0432 (PKC-α
inhibitor) prior to treatment with ZnPP for 24
h. As shown in , SP600125 and Ro 32-0432 had little effect on the activation of StRE3 by ZnPP. SB203580, NAC, and IPA-3 reduced the activation of StRE3 by ZnPP to 72.9%, 62.4%, and 83.6%, respectively, of the level by ZnPP alone. However, LY294002, U0126, and Ro 31-8220 attenuated the activation to 39.4%, 40.2%, and 41.5%, respectively, of the ZnPP-alone control. On the other hand, rottlerin activated StRE3 element by itself and had a synergistic effect with ZnPP (2-fold over the level by ZnPP alone).
Figure 4 Effect of protein kinase inhibitors and antioxidant on ZnPP-activation of StRE3 element of human HO-1 promoter in PC-3 cells. PC-3 cells were transfected with StRE3-pGL3 luciferase reporter plasmid, pretreated with 3μM SB203580, 5 (more ...)
To confirm the effect of LY294002, U0126, Ro 31-8220, and rottlerin on ZnPP-activation of StRE3 element, HO-1 mRNA levels were determined by real-time PCR and protein levels by western blot analyses. For real-time PCR, the relative levels of HO-1 mRNA were determined in cells pretreated with LY294002, U0126, Ro 31-8220, or rottlerin for 1
h prior to ZnPP treatment for 3
h. The results showed that LY294002 and U0126 did not attenuate ZnPP-induction of HO-1 mRNA, but Ro 31-8220 completely suppressed the effect of ZnPP (). On the other hand, real-time PCR also confirmed the synergistic effect of rottlerin with ZnPP; ZnPP alone upregulated HO-1 by 8.2-fold over vehicle-treated control and rottlerin plus ZnPP upregulated HO-1 by 36.0-fold (). Western blot analyses basically confirmed the results of real-time PCR. Ro 31-8220 significantly suppressed the upregulation of HO-1 protein by ZnPP, while LY294002, U0126, and Ro 32-0432 had little effect on ZnPP-induction of HO-1 (). However, the synergistic effect of rottlerin with ZnPP was not evident due to the high level of upregulation of HO-1 by ZnPP alone. Western blot analysis also showed that SB203580 and IPA-3 had no effect on ZnPP-induction of HO-1 expression (data not shown).
Figure 6 Western blot analysis of HO-1 protein induced by ZnPP in PC-3 in the presence of various kinase inhibitors or antioxidant. (a) PC-3 cells were pretreated with nothing (lane 2), 5μM LY294002 (lane 3), 10μM U0126 (lane (more ...)
Since Ro 31-8220 is a pan PKC inhibitor, this suggests that PKC may be involved in ZnPP-upregulation of HO-1. There are many PKC isoforms, but the involvement of PKC-α can be excluded by the lack of suppression of Ro 32-0432 (PKC-α inhibitor) on ZnPP-upregulation of HO-1 protein (, lane 7). To determine if other PKC isoforms may be involved in ZnPP-activation of HO-1, PC-3 cells were treated with myristoylated pseudosubstrates of PKC-θ, PKC-ζ and PKC-η, or PKC-β inhibitor prior to ZnPP treatment. Western blot analysis showed no effect of these inhibitors on HO-1 protein level (). Hence, involvement of PKC-θ, PKC-ζ and PKC-η, and PKC-β can also be excluded.
Since Bach1, Nrf2 and Keap1 proteins have been shown to interact with antioxidant response element, the effect of ZnPP on the mRNA and protein levels of these proteins were investigated by real-time PCR and Western blot analyses. Real-time PCR analyses showed that there were no drastic changes in the expression of BACH1, NFE2L2 (NRF2), and KEAP1 genes when PC-3 cells were treated with ZnPP. The expression levels of BACH1, NFE2L2, and KEAP1 in cells treated with 10μ
M ZnPP for 3
h were 73.5, 81.8, and 98.8%, respectively, of those in control cells. Western blot analysis showed no change in phospho-Nrf2(pS40) protein but a gradual decrease in Keap1 protein in cells treated with ZnPP for 2–24
h (). Only a faint band of Bach1 protein was detected in PC-3 control cells, but none at all in cells treated with ZnPP, despite antibodies from two companies were used. To investigate the effect of ZnPP on the expression of other antioxidant responsive genes, the expression levels of NQO1 and GSTP1 in cells treated with 10μ
M ZnPP for 3
h were determined by real-time PCR. The results showed that the expression levels of NQO1 and GSTP1 were 89.8 and 77.7%, respectively, of those in control cells.
To determine if rottlerin and ZnPP can act as prooxidant, cells were pretreated with 10
mM NAC prior to incubation with 10μ
M ZnPP or 2μ
M rottlerin, and relative levels of HO-1 mRNA were determined by real-time PCR. The results showed that NAC, even at 10
mM, reduced ZnPP-induction of HO-1 expression by only 43% (). Rottlerin by itself did not induce HO-1 mRNA expression after 5
h incubation (), and only a faint band of HO-1 protein was detected on western blot after 24
h incubation with rottlerin (data not shown). Therefore, the effect of NAC on rottlerin was not determined.