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Experimental Biology and Medicine 228:459-465 (2003)
© 2003 Society for Experimental Biology and Medicine

HEME OXYGENASE

Diminished Heme Oxygenase Potentiates Cell Death: Pyrrolidinedithiocarbamate Mediates Oxidative Stress

Lucia Malaguarnera*,1,2, Shuo Quan*, M. Rosaria Pilastro*, Nader G. Abraham{dagger} and Attallah Kappas{dagger}

* Department of Pharmacology, New York Medical College Valhalla, New York 10595 and
{dagger} The Rockefeller University, New York, New York 10021

Abstract

Pyrrolidinedithiocarbamate (PDTC) is a metal-chelating compound that exerts both pro-oxidant and antioxidant effects and is widely used as an antitumor and anti-inflammatory agent. Heme oxygenase-1 (HO-1) is a redox-sensitive-inducible protein that provides efficient cytoprotection against oxidative stress. Because it has been reported that several angiogenic stimulating factors upregulating HO-1 in endothelial cells cause a significant increase in angiogenesis, we investigated the effect of PDTC on cell proliferation and angiogenesis and the effect of overexpression and underexpression of HO-1. The evaluation of PDTC (20 or 50 µM) in endothelial cells resulted in significant increase in HO-1 mRNA and protein (P < 0.001), but a decrease in cell proliferation. Pretreatment of endothelial cells with SnCl2 (10 µM), an inducer of HO-1 attenuated the PDTC-mediated decrease in cell proliferation (P < 0.05). In contrast, pretreatment with SnMP, an inhibitor of HO activity, magnified the inhibiting effect of PDTC on cell proliferation. Upregulation of HO-1 gene expression by retrovirus-mediated delivery of the human HO-1 gene also attenuated the PDTC-induced decrease in cell proliferation. Underexpression of HO-1, by delivery of the human HO-1 in antisense orientation, enhanced the PDTC-mediated decrease in cell proliferation. The decrease, by PDTC, in proliferation of cells underexpressing HO-1 is related to an increase in O-2 production. Collectively, these results demonstrate that upregulation of HO-1 was able to attenuate the PDTC-mediated cell proliferation, but was unable to reverse the high concentration of PDTC-induced decrease in angiogenesis.

Key Words: oxidative stress • angiogenesis • cell proliferation

Pyrrolidinedithiocarbamate (PDTC) belongs to the group of dithiocarbamates, which are metal chelating compounds reported to exert numerous effects in biological systems. Dithiocarbamates have been used clinically in the treatment of pathogenic fungi and bacteria (1), in chelation therapy for nickel and copper poisoning, and in the experimental treatment of AIDS (2). PDTC is a widespread pharmacological agent in molecular and cell biology, where it is reported to have both pro-oxidant and antioxidant properties (3). It has also been shown that PDTC can inhibit apoptosis in lymphocytes (4), neurons (5), and vascular and endothelial cells (6). Other recent studies have suggested the possibility that PDTC promotes oxidation of glutathione, markedly stimulating apoptosis in thymocytes, an activity that is attributed to the capacity of PDTC to exert a pro-oxidant effect by increasing the intracellular level of redox-active copper (3). Other studies have shown that apoptosis is also induced by PDTC in vascular smooth muscle cells (7), in mature rabbit osteoclasts (8), and in murine B cells (9). Recently, it has been demonstrated that PDTC is a potent inducer of the stress-inducible gene, heme oxygenase-1 (HO-1)(0).

HO-1, the initial and rate-limiting enzyme in heme catabolism, is a stress protein that plays a central role in diverse biological systems, including cell respiration, energy generation, oxidative biotransformation, growth differentiation processes, and generation of inflammatory mediators, such as nitric oxide and eicosanoides (11, 12). The induction of HO-1 is an essential step in the cellular adaptation to stress inflicted by pathological events. Conversely, deficient HO-1 expression in mammalian cells contributes to a reduced stress defense (13, 14). Recently, it has been shown that HO-1 induction prevents oxidant-stressed endothelial upregulation of adhesion molecules and the development of transplant artheriosclerosis in normal mice (13, 14). We have previously demonstrated that overexpression of HO-1 in coronary endothelial cells resulted in resistance against heme-hemoglobin-induced injury (15, 16), demonstrating that moderate increases in HO-1 activity is beneficial in resistance to oxidants.

One key effect seems to be the ability of HO-1 to degrade the intracellular pro-oxidant heme (15, 16) by generating bilirubin and carbon monoxide (CO), both of which play an important role in cellular antioxidative reactions. Bilirubin acts as a potent peroxyl radical scavenger (17–19). A wide body of evidence has demonstrated that increased CO and biliverdin contribute to modulate important physiological processes within the cardiovascular, immune and nervous systems.

HO-1 is increased in whole animal tissues and in cultured cells after treatment with heme, metals, and inflammatory cytokines as well as in hypoxic and oxidative conditions. It is well known that HO-1 can be induced by an imbalance in the redox status of thiols after a challenge with oxidants (18). This is confirmed by in vitro and in vivo evidence showing that, in several stress-related circumstances, stimulation of HO-1 is directly associated with a change in intracellular glutathione levels (20). The evidence that HO-1 expression can be regulated by redox signaling events is also confirmed by data showing that transcription of this gene is suppressed by thiols and certain antioxidant (21). Although the key factors participating in signal transduction mechanisms and the specific chemical modifications required for transcriptional activation of HO-1 remain to be fully identified, this enzyme can be regarded as a potential therapeutic target in a variety of oxidant- and inflammatory-mediated diseases. In this regard, the search for novel and more potent inducers of this pathway will facilitate the development of pharmacological strategies to increase the intrinsic capacity of cells to maximize HO-1 expression and, consequently, promote cytoprotection. More recently, transcriptional regulation of HO-1 mRNA levels have been seen with PDTC (10). Previously, we demonstrated that overexpression of the HO-1 gene in endothelial cells caused a significant increase in angiogenesis (22), somatic cell growth and cell proliferation (23, 24).

Our goal in this study was to investigate whether PDTC, a potent inducer of the stress-inducible gene, HO-1, modulates cell survival and angiogenetic effects in human endothelial cells. Our data show for the first time that, although upregulation of the HO gene is an important defense mechanism to protect the cells against peroxidative stress and attenuates superoxide anion (O-2), cell death, However, the angiogenetic effect of HO-1 was inhibited by higher concentrations of PDTC.

Materials and Methods

Cell Culture Conditions.
The human dermal microvessel endothelial cells transduced with the human HO-1 gene in sense and antisense orientations used in this study were a kind gift of Nader G. Abraham (New York Medical College, Valhalla, NY). All cells were incubated at 37°C in a 5% CO2 humidified atmosphere, and maintained at subconfluency by passaging with trypsin-EDTA.

Northern Blot.
Total RNA was extracted with the use of Trizol reagent (GIBCO BRL) according to the manufacturer’s instructions. Total RNA (10 µg/lane) from PDTC-treated and untreated samples was denatured, electrophoresed on 1.2% agarose formaldehyde gels, transferred to a positively charged nylon membrane (Hybond N+; Amersham Inc., Piscataway, NJ), and UV cross-linked (Stratalynker; Stratagene, La Jolla, CA). Membranes were prehybridized for 1–2 hr at 60°C and were subsequently hybridized overnight at 60°C with random primer (GIBCO BRL) 32P dCTP-labeled HO-1. The blots were washed three times with a solution containing 0.5% BSA, 5% SDS, and 1 mM EDTA in 0.2 saline sodium citrate at 56–60°C and then were exposed to x-ray film at -80°C.

Western Blot and HO Activity.
Cells were harvested using cell lysis buffer as described previously (25). The lysate was collected for Western blot analysis. Protein levels were visualized by immunoblotting with antibodies against human HO-1, total immunoreaction HO-1 (rat and human) or HO-2. HO activity was determined by methods of Abraham et al. (25).

Cell Proliferation.
Cells were seeded in 96-well culture plates (4 x 104) and grown for 24 hr. Subconfluent cells were then treated with different concentrations of PDTC (20, 50, 100 µM/ml). Cells were divided to several groups, and the groups were pretreated with an inducer of HO-1 activity, stannic chloride (SnCl2, 10 µM), or with the inhibitor, stannic mesoporphyrin (SnMP, 10 µM) for 8 h, and then with PDTC. Cell proliferation was conducted using a 5-bromo-2' deoxy-uridine colorimetric kit (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer’s instructions.

Superoxide Anion (O-2) Production.
The O-2 production was assayed by the spectrophotometric measurement of ferricytochrome c reduction. Cells were harvested 4 hr after PDTC treatment. Cells were washed once with PBS and incubated with 0.5 ml of reaction mixture consisting of Krebs Ringer phosphate buffer containing 80 µM cytochrome c, 2 mM NaN3. After 1 hr of incubation at 37°C, the supernatants were collected and used to assay the amount of reduced cytochrome c by the difference in adsorbance at 550–468 nm. The O-2 release was calculated using a coefficient of 0.0245 (the extinction coefficient µml/l of Cytochrome c determined at 550–468 nm), and expressed as µmol O-2/mg protein.

In Vitro Angiogenesis and Capillary Formation.
A growth factor induced basement membrane Matrigel matrix was used for assessment of in vitro capillary formation as previously described (22).

Results

HO-1 Expression in Endothelial Cells after PDTC Treatment.
We evaluated the effect of PDTC on HO-1 gene expression by measuring HO-1 mRNA levels. Total mRNA of endothelial cells treated with different concentrations of PDTC (20 or 50 µM/ml) for 2 hr was subjected to Northern blot analysis and probed for HO-1 and G3PDH. As seen in Figure 1Go, the levels of HO-1 mRNA in endothelial cells treated with 20 and 50 µM/ml of PDTC were elevated compared with the transcript levels in untreated cells (Fig. 1Go, Lanes 1, 2, and 3, respectively). Hybridization of the filters with a radiolabeled G3PDH probe confirmed that a similar amount of total mRNA was transferred to the filters in each lane of the paired experiment (Fig. 1Go, lower panel). Quantitative evaluation of HO-1 mRNA changes by densitometry analysis indicated a 3.8- and 3.6-fold increase in HO-1 mRNA levels in endothelial cells treated with 20 and 50 µM PDTC, respectively, for 2 hr compared with untreated controls.



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  		Figure 1. Northern blot analysis of HO-1 mRNA expression from endothelial cells untreated and treated for 2 hr with different concentrations of PDTC. Lane 1, control; Lane 2, PDTC 20 µM/ml; Lane 3,) PDTC 50 µM/ml.

 
Enhancement of HO-1 Synthesis in Endothelial Cells after PDTC Exposure.
To assess whether the increase in HO-1 mRNA was associated with the elevation in HO-1 protein, Western blot analysis was performed on cell lysates obtained from PDTC treated and untreated endothelial cells. Using HO antibodies that recognize both human and rat HO-1 protein, we demonstrated a dose-dependent increase in HO-1 protein. Figure 2Go shows HO-1 protein synthesis following exposure to 20 and 50 µM PDTC. A strong signal for HO-1 is evident in Figure 2Go, Lanes 2 and 3. Cells treated with 10 µM SnMP were used as control. A similar amount of HO-2 and ß-actin proteins were observed in the presence or absence of PDTC. Quantitative evaluation of HO-1 protein was measured by scanning densitometry. The results showed that HO-1 synthesis was increased 3.7- and 4.0-fold over control within 4 hr of treatment with 20 µM and 50 µM, respectively. These results confirm that HO-1 enhancement is a mechanism that cells use to protect themselves from PDTC action.



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  		Figure 2. (A) Western blot analysis of endothelial cells untreated and treated for 4 hr with different concentrations of PDTC. Lane 1, control; Lane 2, PDTC 20 µM/ml; Lane 3, PDTC 50 µM/ml; Lane 4, Heme 10 µM. (B) Levels of HO-1 protein normalized to HO-2.

 
Effect of PDTC on Endothelial Cell Proliferation.
Proliferation of endothelial cells is a critical indicator of the effect of oxidant-mediated injury. To verify the effect of PDTC in endothelial cell proliferation, cells were incubated with different concentrations of PDTC (20, 50, 100 µM/ml). Cell number was counted kinetically at 24 hr. As shown in Figure 3Go, 20 and 50 µM PDTC caused a significant decrease in cell proliferation, and a dramatic decrease in cell number was observed with 100 µM/ml PDTC. To assess whether PDTC counteracted HO-1 activity, it was essential to evaluate the effect of inducers (SnCl2, 10 µM) and inhibitors (SnMP, 10 µM) on HO-1 activity. As seen in Figure 3Go, pretreatment with SnCl2 followed by treatment with 20 µM of PDTC resulted in a slight increase in endothelial cell proliferation compared with the effect of PDTC alone. In contrast, SnMP pretreatment increased the inhibition of cell proliferation that PDTC induced, suggesting that the expression of HO-1 plays a significant role in cellular resistance to oxidants.



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  		Figure 3. Dose-response effects on cell proliferation of human microvessel endothelial cells treated with different concentrations of PDTC (20, 50, 100 µM/ml) for 24 hr in 0.5% of fetal bovine serum and pretreated for 8 hr with and SnCl2 (10 µM), and SnMP (10 µM) and then incubated with PDTC 20 µM/ml for 24 hr in 0.5% of fetal bovine serum. Data are representative of three independent experiments. Cell proliferation was measured as described in Materials and Methods.

 
Effect of PDTC on Endothelial Cell Proliferation in HO-1 Sense and HO-1 Antisense.
We next examined the proliferative effect of PDTC in human endothelial cells transduced with the retroviral HO-1 construct in sense and HO-1 antisense orientation sequences. As seen in Figure 4Go, at 20 µM/ml, PDTC was ineffective in cells overexpressing HO-1 in the sense orientation. In contrast, PDTC produced a decrease in cell proliferation in cells overexepressing HO-1 in the antisense orientation compared with control cells (P < 0.05). This finding suggests that HO-1 overexpression protects endothelial cells from the cytotoxic effect of PDTC.



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  		Figure 4. Effects on proliferation of human microvessel endothelial cells: control, HO-1 sense and HO-1 antisense treated with PDTC (20 µM/ml). Data are representative of three independent experiments. Cell proliferation was measured as described in Materials and Methods.

 
In Vitro Angiogenesis and Capillary Endothelial Cell Formation Induced by PDTC.
To assess the effect of PDTC on angiogenesis, formation of branching and anastomosing capillary-like cords were experimentally induced by embedding endothelial cells within a Matrigel matrix in the presence or absence of PDTC 50 µM/ml. The amount of tubular growth in control cells was significantly decreased in the presence of PDTC and the tubules were also less pronounced, shorter and less numerous (Fig. 5AGo) but in cells overexpressing HO-1, the of tubular growth were more than that in presence of 25 µM PDTC (Fig. 5BGo), but was unable to reverse the high concentration of 50 µM PDTC-induced decrease in angiogenesis (data not shown).



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  		Figure 5. In vitro angiogenesis of cells: (a) control; (b) cells treated with PDTC. Quantitative analysis was performed as described in Materials and Methods. Data plotted are given in the lower panel. Results are presented as percentage increase in vessel tube size over control.

 
Superoxide Anion (O-2) Production in Endothelial Cells.
To investigate the mechanisms by which PDTC exerts an apoptotic effect in endothelial cells, we measured O-2 production by the spectrophotometric measurement of ferricytochrome c reduction. PDTC induced O-2 production in a dose-dependent manner. At 50 µM, production of O-2 was increased 3.3-fold over that of the control within 4 hr of PDTC treatment (Fig. 6Go). We also tested O-2 production in endothelial cells transduced with the retroviral HO-1 constructed in sense and in antisense orientation sequences. PDTC significantly increased O-2 production in cells overexpressing HO-1 in the antisense orientation. In contrast, PDTC was unable to induce O-2 production in cells overexpressing HO-1 in the sense orientation (data not shown). These findings suggest that the sustained elevation of oxidative stress in endothelial cells may be responsible for PDTC-induced apoptosis.



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  		Figure 6. Effect of PDTC (20 or 50 µM) on superoxide anion production in human microvessel endothelial cells in HO-sense and HO-antisense cells. All results are expressed as mean ± SD of three independent determinations.

 
Discussion

In the present study, we demonstrated that, at certain concentrations, PDTC significantly increased HO-1 gene expression in endothelial cells. Although upregulation of the HO gene is known to enhance endothelial cell growth and angiogenesis, the angiogenetic effect of HO-1 was inhibited by PDTC. These results clearly indicate that PDTC significantly decreased cell proliferation in a dose-dependent manner. Pretreatment with SnCl2, an inducer of HO-1, followed by treatment with PDTC resulted in a slight increase of endothelial cell proliferation compared to the effect of PDTC alone. This finding suggests that HO-1 protects the cells from the cytotoxic effect of PDTC. To further explore the protective role of HO-1 against oxidative stress caused by PDTC, we used the retroviral-mediated delivery of the HO-1 gene as recently described (24), in which cells transduced with the HO-1 gene in sense and antisense orientations resulted in an increase or decrease in HO-1 expression, respectively. PDTC is also a proapoptotic compound through activation of caspase. When PDTC was added to cells overexpressing HO-1 mRNA and protein, HO-1 produced a significant inhibition of caspase 3 activity (data not shown). The results from the caspase data (not shown) and proliferation assays showed that the HO-1 gene transduced endothelial cells could resist higher doses of PDTC compared with nontransduced cells. These data corroborate previous evidence that overexpression of HO-1 plays a crucial role in the cellular protective system (15, 26). We, and others, have previously shown that upregulation of HO-1 enhances angiogenesis (22, 23, 27). In this study, we detected the effect of PDTC on capillary formation and provide direct evidence that HO-1 upregulation is unable to counteract the antiangiogenetic effect of PDTC.

Persistent oxidant damage, caused by increased production of free radical species along with recurrent inflammation, characterizes the development of numerous pathologies, including vascular dysfunction and carcinogenesis. Mammalian cells have developed highly inducible systems, against the generation of intracellular toxic mediators, which can be engaged to alleviate and hinder the manifestation of a distinctive metabolic disorder. HO-1 has a pivotal role in the resolution of acute inflammatory states as well as in the protection against oxidative damage and nitrosative stress. Our study as well as other studies (28, 29) strongly support the hypothesis that HO-1 gene induction is essential to restore cellular homeostasis and that the beneficial effects of increased heme oxygenase activity may represent a promising therapeutic expedient to preclude tissue injury and impede the progression of a variety of diseases. The efficacy of HO-1 in promoting cytoprotection resides in the ability of its metabolic products, such as CO and bilirubin, to exert potent antioxidant and anti-inflammatory activity (30). Recently, it has been demonstrated that PDTC attenuates the development of acute and chronic inflammation (31). Our findings show that PDTC-induced HO-1 overexpression acts to protect endothelial cells against oxidative stress. It is known that PDTC is a specific inhibitor of nuclear factor-{kappa}B (32). This compound also exerts an antitumorigenic effect in many different cancers (33, 34). Our studies reveal a potential novel aspect in the mode of action of PDTC, although through the stimulation of the HO-1 pathway, it can be counted as a potent antiangiogenetic compound. Once added to endothelial cells, the potency of PDTC in increasing HO-1 expression appears to be strictly associated with a rapid change in the intracellular redox status. PDTC is well known for its ability to bind free or protein-bound metals, and exerts both antioxidant and pro-oxidant effects in cells. As an antioxidant, PDTC eliminates hydrogen peroxide and scavenges the superoxide radical, peroxinitrite, the hydroxyl radical, and lipid peroxidation products such as the peroxyl radical.

Our results demonstrate that, in the early stages of the treatment with PDTC, a significant loss in cell viability was associated with an increase in the superoxide anion content. This result is consistent with the notion that changes in the redox status of the cell are a prerequisite for the upregulation cytoprotective genes, such as HO-1, and that a more severe oxidation results in suppression of the cellular stress response, ultimately leading to cell death. Moreover, suppression of HO activity by SnMP or inhibition of HO-1 expression by antisense oligonucleotides abolished the protective effect of pre-irradiation, further implicating HO-1 in the mechanism of cellular protection.

In conclusion, PDTC exerts its effects in a concentration-dependent manner. Based on its pro-oxidant and proapoptotic properties, we have identified PDTC as a promising anti-angiogenetic compound having not been tested yet in anti-agiogenetic therapy for tumor growth or chronic inflammatory diseases. Because the HO-1 gene is stimulated at transcriptional levels, PDTC would offer a great advantage in therapeutic trials for protection against oxidative stress and simultaneously PDTC-inhibited angiogenesis.

Footnotes

1 Current address: University of Catania, Via Androne, 83, Department of Biomedical Sciences, Catania, Italy. Back

2 To whom requests for reprints should be addressed at University of Catania, Via Androne, 83, Department of Biomedical Sciences, Catania, Italy. Back

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Pharmacological and Clinical Aspects of Heme Oxygenase
Pharmacol. Rev., March 1, 2008; 60(1): 79 - 127.
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