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Cancer Research 67, 7301, August 1, 2007. doi: 10.1158/0008-5472.CAN-06-4010
© 2007 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Protection of Zinc against Tumor Necrosis Factor–Induced Lethal Inflammation Depends on Heat Shock Protein 70 and Allows Safe Antitumor Therapy

Wim Van Molle1,2, Maarten Van Roy1,2, Tom Van Bogaert1,2, Lien Dejager1,2, Philippe Van Lint1,2, Ineke Vanlaere1,2, Kenji Sekikawa3, George Kollias4 and Claude Libert1,2

1 Department for Molecular Biomedical Research, VIB; 2 Department of Molecular Biology, Ghent University, Ghent, Belgium; 3 Department of Molecular Biology and Immunology, National Institute of Agrobiological Sciences, Ibaraki, Japan; and 4 Institute of Immunology, Biological Sciences Research Center Alexander Fleming, Vari, Greece

Requests for reprints: Claude Libert, Department for Molecular Biomedical Research, VIB, Technologiepark 927, B-9052 Ghent, Belgium. Phone: 32-9-331-3700; Fax: 32-9-331-3609; E-mail: Claude.Libert{at}dmbr.UGent.be.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tumor necrosis factor (TNF)–induced inflammation prevents its broad application as an antitumor agent. We here report that addition of ZnSO4 to the drinking water of mice induces expression of heat shock protein 70 (HSP70) in several organs, notably the gastrointestinal track. Zinc conferred dose-responsive protection against TNF-induced hypothermia, systemic induction of interleukin-6 and NOx, as well as against TNF-induced bowel cell death and death of the organism. The protective effect of zinc was completely absent in mice deficient in the major HSP70-inducible gene, hsp70.1, whereas transgenic mice constitutively expressing the human HSP70.A gene, under control of a ß-actin promoter, was also protected against TNF, indicating that an increase in HSP70 is necessary and sufficient to confer protection. The therapeutic potential of the protection induced by ZnSO4 was clearly shown in a TNF/IFN{gamma}–based antitumor therapy using three different tumor models. In hsp70.1 wild-type mice, but not in hsp70.1-deficient mice, zinc very significantly protected against lethality but left the antitumor effect intact. We conclude that zinc protects against TNF in a HSP70-dependent way and that protection by zinc could be helpful in developing a safer anticancer therapy with TNF/IFN{gamma}. [Cancer Res 2007;67(15):7301–7]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The cytokine tumor necrosis factor (TNF) was named after its impressive antitumor activity (1). Indeed, TNF, especially in combination with IFN{gamma}, can destroy tumors in experimental animals and human patients (2). Unfortunately, TNF is also a proinflammatory cytokine whose injection leads to inflammation, hypotension, liver toxicity, and bowel necrosis (3–5). This effect hampers the systemic use of TNF in cancer patients. Therefore, the use of TNF is restricted to regional treatment (6, 7). Moreover, TNF has also been shown to be involved in important diseases, such as rheumatoid arthritis and inflammatory bowel disease (IBD; refs. 8, 9). Inhibition of the toxicity of TNF is thought to be important to broadening its value as a therapy for cancer and to protect against arthritis and IBD. We have reported recently that whole-body heat shock (WBHS) of mice led to the induction of heat shock protein 70 (HSP70) and to complete protection against TNF-induced lethality. This protection depended on HSP70 (5). In our search for other inducers of HSP70, we studied the effects of heavy metals. Most heavy metals are toxic, but some, such, as zinc, are safe to use. Zinc is used to treat the common cold, rhinitis, pneumonia, and dermatitis (10–14) and zinc has been shown to protect in pig and rat models of endotoxemia (15, 16). We here show that addition of zinc to the drinking water of mice protected them against TNF-induced lethal shock, metabolic disturbances, and bowel toxicity. Zinc-induced HSP70 and the protection depended on the induced HSP70. The protection conferred by zinc is so robust that it improves survival of tumor-bearing mice during therapy with TNF and IFN{gamma}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice. Female C57BL/6 mice were from Iffa-Credo and used at the age of 8 to 10 weeks. Hsp70.1-deficient mice (C57BL/6 background) were generated as described previously (5) and bred as homozygotes in our facilities. Hsp70tg mice, expressing the human hsp70.A gene under control of a ß-actin promoter, were generated as reported (17). Wild-type (WT) controls had an identical genetic background (17). Breeding couples were kindly provided by Dr. G. Pagoulatos. All experiments were approved by the ethics committee of the Faculty of Sciences of Ghent University.

Reagents. Recombinant mouse TNF and IFN{gamma} were produced in Escherichia coli and purified to homogeneity in our laboratories. TNF and IFN{gamma} had specific activities of 1.2 x 108 and 1.16 x 107 IU/mg, respectively, with no detectable endotoxin contamination.

Injections and blood collection. Cytokines and reagents were diluted in lipopolysaccharide (LPS)–free PBS before injecting them s.c. (0.1 mL), i.v. (0.2 mL), or i.p. (0.5 mL). Blood was withdrawn from the retro-orbital plexus and allowed to clot for 30 min at 37°C and overnight at 4°C, and the clot was removed and cells were pelleted by centrifugation at 20,000 x g for 10 min. Serum was prepared and stored at –20°C.

Tumor cell culture and inoculation. B16BL6, PG19, and LLC cells were cultured in DMEM supplemented with FCS, antibiotics, and L-glutamine. Cells were harvested, washed thrice in LPS-free PBS, and brought to a concentration of 6 x 106/mL (B16BL6) or 50 x 106/mL (PG19 and LLC), of which 100 µL were injected s.c. into the right hind limbs of the mice.

Tumor size index and body temperature. The smaller and larger diameters of the tumors were measured with an electronic caliper, and tumor size index (TSI) was calculated by multiplying these two values. Rectal body temperature was determined with an electronic thermometer (model 2001, Comark Electronics).

WBHS induction. A 20-min WBHS was induced as published (5, 18–20).

Tissue embedding, tissue sectioning, and staining. Mice were euthanized and tissues were removed and fixed in 4% paraformaldehyde. Tissues were passed through baths of 50%, 70%, 95%, and 100% ethanol and 100% Histo-clear, embedded in paraffin, and cut at 4 µm with a microtome. The sections were stained with H&E. Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) staining was done with the DeadEnd colorimetric apoptosis detection system (Promega Biotec) as described earlier (5).

Bioassays and detection of caspase activity. Serum interleukin-6 (IL-6) was determined with a 7TD1 bioassay (21). Serum nitrate and nitrite levels (NOx–) were assessed essentially as described (22). Caspase-like activities in bowel samples were determined using an acetyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-aminomethyl-coumarin (Ac-DEVD-amc) system as described previously (5).

Western blot. Total proteins (50 µg) of the different tissue homogenates were separated in a 7.5% polyacrylamide gel and blotted on a nitrocellulose membrane (Schleicher & Schuell). Blots were blocked with 1% bovine serum albumin (BSA) and 0.1% Triton X-100 overnight at 4°C. They were incubated for 2 h at room temperature with a biotinylated mouse anti-HSP70 antibody (Stressgen Biotechnologies Corp.), diluted 1:3,000 in BSA/Triton X-100. After three washes, blots were incubated with streptavidin-AP (1:1,500; BioSource International) and developed with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Roche Molecular Biochemicals).

Statistical analysis. Survival curves (Kaplan-Meier plots) were compared using a log-rank test. Final mortality (deaths/total) were compared with a {chi}2 test. Means ± SD were compared with a Student's t test. *, **, and *** represent 0.01 < P < 0.05, 0.001 < P < 0.01, and P < 0.001, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Zinc induces HSP70 and protects mice against TNF-induced lethality. To document the induction pattern of HSP70 by ZnSO4, mice received water or 25 mmol/L ZnSO4 and sacrificed 7 days later. The expression of HSP70 in liver, lung, jejunum, and colon was studied by Western blotting. In mice receiving normal water, some minimal background expression of HSP70 in liver, lung, and jejunum was observed, as well as clear constitutive expression in the colon. Addition of ZnSO4 to the drinking water led to increased expression in liver and jejunum. On the other hand, expression in lung remained rather low and expression in colon did not change (Fig. 1A ).


Figure 1
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  		Figure 1. ZnSO4 induces HSP70, protects against TNF-induced mortality, and prevents release of NO and IL-6. A, mice received normal drinking water or 25 mmol/L ZnSO4 and were sacrificed 7 days later. Liver, lung, jejunum, and colon were removed, and HSP70 expression was visualized by Western blot. Top, HSP70; bottom, actin. B and C, mice received normal drinking water (n = 15; {circ}), 5 mmol/L ZnSO4 (n = 15; bullet), 12.5 mmol/L ZnSO4 (n = 15; {square}) or 25 mmol/L ZnSO4 (n = 15; {blacksquare}), and 7 days later, they were challenged i.v. with 12.5 µg TNF. Mortality and body temperature were scored at several time points until no further deaths occurred. D and E, mice received normal drinking water (n = 36; {circ}) or 25 mmol/L ZnSO4 (n = 36; bullet), and 7 days later, they were challenged i.v. with 12.5 µg TNF. Blood was withdrawn and analyzed for serum concentrations of NOx– (D) and IL-6 (E) at time points 1, 3, 6, 9, 12, and 24 h after injection (n = 6 per time point, unless otherwise indicated).

 
To study the protective effect of ZnSO4 against TNF, we did a dose response experiment. Mice received increasing doses of ZnSO4 in the drinking water (n = 15 for each group), and 7 days later, they were challenged i.v. with 12.5 µg TNF (i.e., a LD100 dose). Mortality was scored up to 72 h after the challenge. We show (Fig. 1B) that ZnSO4 conferred significant and dose-dependent protection against TNF-induced mortality compared with control mice. Although all three doses of ZnSO4 provided significant delay of death (P = 0.0209, P = 0.0003, and P < 0.0001 for 5, 12.5, and 25 mmol/L, respectively), the best protection in terms of final survival rate was observed with the highest dose of ZnSO4 (13 of 15 survival for 25 mmol/L ZnSO4 versus 0 of 15 for controls; P < 0.0001). Increasing the concentration of ZnSO4 had no additional protective effect.

To investigate the effects of ZnSO4/HSP70 on TNF-induced nitric oxide (NO) and IL-6, mice received 25 mmol/L ZnSO4 (n = 36) or water (n = 36), and 7 days later, they were challenged i.v. with 12.5 µg TNF (LD100). One, 3, 6, 9, 12, and 24 h after the challenge, blood was withdrawn and serum was prepared for nitrate/nitrite and IL-6 determinations. ZnSO4 significantly inhibited NO production 9 h (P = 0.0163), 12 h (P = 0.0009), and 24 h (P = 0.0005) after the challenge compared with the corresponding control groups (Fig. 1C). It also reduced the levels of TNF-induced IL-6 after the challenge (9 h, P = 0.034; 12 h, P = 0.0012; and 24 h, P = 0.0002; Fig. 1D). These data show that ZnSO4 significantly blocks TNF-induced NO and IL-6 release, especially at the later time points, leading to transient rather than sustained expression profiles.

When mice are treated with high doses of TNF, rapid swelling of the intestine and epithelial damage are observed. This extremely heavy damage supposedly plays an important role in lethality because it may lead to influx of gut flora (4, 5). To evaluate whether ZnSO4 prevents TNF-induced damage of the bowel, mice received normal drinking water or ZnSO4 (n = 12), and 7 days later, they were challenged with TNF (LD100). One hour after the challenge, the whole small intestine (duodenum, jejunum, and ileum) was clamped, removed, and weighed with its content. We show (Fig. 2A ) that the weight of the small intestine of TNF-treated mice was significantly higher than that of PBS-treated controls (P < 0.0001) and that ZnSO4 resulted in a significant inhibition of the observed swelling (P = 0.0002). Part of the jejunum was then isolated and homogenized, and the cleavage activity of the chromogenic substrate Ac-DEVD-amc was determined as a measure of the activity of caspase-3 and caspase-7, both known as executioner caspases. In jejunum homogenates of mice challenged with TNF, we found a significant increase in DEVD cleavage activity compared with homogenates from PBS-treated mice (P = 0.0091). This increase was significantly inhibited when TNF-challenged mice were pretreated with ZnSO4 compared with TNF treatment (P = 0.0467; Fig. 2A). Samples of the jejunum stained with H&E showed that treatment with TNF (Fig. 2B) resulted in severe damage to the jejunum compared with untreated animals (Fig. 2B). Villi are flattened because their distal parts were eroded and denuded at the top. The crypts are distended and contain mucus and debris. Pretreatment of mice with ZnSO4 markedly reduced the damage (Fig. 2B): shortening of the villi is less prominent and the crypts retain their normal architecture. Tissue sections were also analyzed by TUNEL assay to stain apoptotic cells. In control mice, no TUNEL staining was observed (Fig. 2B). In contrast, the tops of the villi of TNF-treated mice show positive TUNEL staining in the form of brown nuclear staining (Fig. 2B). In mice receiving ZnSO4 in the drinking water, TUNEL staining after TNF was nearly completely absent (Fig. 2B). These data show that ZnSO4 completely prevents the TNF-induced tissue damage and apoptosis in the jejunum.


Figure 2
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  		Figure 2. ZnSO4 prevents TNF-induced bowel necrosis. Mice received normal drinking water or 25 mmol/L ZnSO4, and 7 days later, they were challenged i.v. with TNF. A, 1 h after the challenge, the entire small intestine was isolated and weighed with its contents. Parts of the jejunum were homogenized for assessment of DEVDase activity. B, other parts were embedded in paraffin and stained with H&E or processed for TUNEL assay. Bar, 10 µm.

 
Zinc does not protect in HSP70.1-deficient mice. Next, we studied whether the protection by ZnSO4 is due to the induction of HSP70 and therefore studied the effects of ZnSO4 in hsp70.1–/– mice. These mutant mice are as sensitive to TNF-induced mortality as WT animals. hsp70.1–/– mice received normal drinking water (n = 11) or 25 mmol/L ZnSO4 (n = 12), and 7 days later, they were challenged with 10 µg TNF. As positive controls, WT mice received normal drinking water (n = 11) or 25 mmol/L ZnSO4 (n = 13) and were challenged with TNF. The induction of HSP70 by ZnSO4 is clear in WT control mice but severely reduced in the hsp70.1–/– mice (data not shown). We observed some HSP70 induced by ZnSO4 in the hsp70.1–/– mice, but this signal probably results from induction of the other inducible HSP70 gene, hsp70.3, which is located in the same locus as hsp70.1, and which is still present in the hsp70.1–/– mice. The genes and protein sequences of HSP70.1 and HSP70.3 are identical; hence, both are detected with our antibody. By contrast, their respective promoter regions are different and they are controlled in a different manner (5). The residual HSP70 does not seem to be sufficient to confer some protection against TNF. Indeed, WT mice were very significantly protected (P < 0.0001; Fig. 3A ), but no protection whatsoever was observed in the hsp70.1–/– mice (Fig. 3B). Furthermore, we observed that ZnSO4 significantly protected against TNF-induced hypothermia (P < 0.0001), NO (P < 0.0001), and IL-6 (P = 0.0036) in WT mice (measured 12 h after challenge), but not in the hsp70.1–/– mice (all variables P > 0.05; Fig. 3C and D). These data show that all ZnSO4-mediated protective effects against TNF require the induction of HSP70.1 and that HSP70.1 is needed for the ZnSO4-induced inhibition of TNF-induced gene induction.


Figure 3
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  		Figure 3. ZnSO4-mediated protection is absent in hsp70.1–/– mice. WT mice (n = 13; bullet) and hsp70.1–/– (n = 14; {blacksquare}) mice received 25 mmol/L ZnSO4 and were challenged 7 days later with 10 µg TNF. WT (n = 11; {circ}) and hsp70.1–/– (n = 11; {square}) mice on normal drinking water were also challenged with 10 µg TNF. Twelve hours after injection, body temperature was recorded and blood was withdrawn for serum preparation. A and B, survival rate, no further deaths occurred. C and D, body temperature, serum NOx–, and IL-6 concentrations.

 
HSP70 transgenic mice are protected against TNF. The hsp70tg (TG) mice express the human inducible hsp70.A gene under control of the ß-actin promoter and exhibit improved recovery in a model of cardiac ischemia and reperfusion injury (7). We studied the basal expression pattern of HSP70 in TG mice and observed increased HSP70 expression in several organs compared with isogenic WT controls (Fig. 4A ). Next, we injected WT and TG mice with 12.5 µg TNF, a dose that was 100% lethal for the WT mice (14 of 14), but only for 1 of 16 TG mice (P < 0.0001; Fig. 4B). The TG mice displayed a significantly reduced TNF-induced hypothermia (P = 0.0049), NO (P = 0.0001), and IL-6 (P = 0.0009), as measured 12 h after challenge (Fig. 4C).


Figure 4
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  		Figure 4. Hsp70 TG mice are resistant to TNF-induced mortality. A, naive WT and TG mice were sacrificed and several organs were removed and homogenized. HSP70 expression was visualized by Western blotting. Top, HSP70; bottom, control for equal protein loading. B and C, WT (n = 14; {circ}) and TG (n = 16; bullet) mice were challenged i.v. with 12.5 µg TNF and survival was recorded. Twelve hours after the challenge, body temperature was measured and blood was withdrawn to analyze serum NOx– and IL-6 concentrations.

 
Zinc and WBHS protect against TNF/IFN{gamma} in three different tumor models. Three different C57BL/6 syngeneic tumor models were used in the following study. C57BL/6 mice were inoculated with a B16BL6 melanoma, a PG19 melanoma, or LLC tumor. Application of TNF together with IFN{gamma} induces regression of these tumors, but it is accompanied with high mortality (2). To reduce or inhibit this toxicity, we evaluated the application of ZnSO4 or WBHS induction in an antitumor protocol. WBHS had been shown to protect in the B16BL6 model before (5).

In all three antitumor experiments, mice were inoculated s.c. with tumor cells on day 0 and divided randomly into six groups on day 3. One group (n = 8) received daily s.c. injections of PBS for 10 days (days 11–20). The second group (n = 8) received daily WBHS for 10 days (days 11–20). The third group (n = 8) was treated with 25 mmol/L ZnSO4 in the drinking water starting from day 3 to day 19. The other three groups (n = 10) were treated as the three first groups but received daily s.c. injections of 10 µg TNF in combination with 5,000 IU IFN{gamma} for 10 consecutive days (days 11–20). TNF/IFN{gamma} was given 12 h after WBHS. The combination of TNF and IFN{gamma} was administered s.c. near the tumor and not i.v. because the former application allows the best tumor regression. The TSI was recorded from day 10 (start treatment) until day 19 (stop treatment). Mortality was monitored for 1 week after the last day of treatment (no further deaths occurred). In the three tumor models, mice treated with PBS, WBHS, or ZnSO4 displayed rapid tumor growth, whereas TNF/IFN{gamma} treatment led to complete regression of the tumor. Neither WBHS nor ZnSO4 pretreatment had any effect on the antitumor effect of TNF/IFN{gamma} (Fig. 5A, C, and E ). When evaluating the toxicity of the treatment, it became clear that the dose of TNF/IFN{gamma} in all three tumor models was very toxic, leading to death of all the B16BL6 and PPG19 mice and to 9 of 10 of the LLC mice (Fig. 5B, D, and F). In all three models, both WBHS and ZnSO4 had a very significant protective effect against death from the TNF/IFN{gamma} treatment (see Fig. 5 for P values). In the B16BL6 model, zinc was more effective than WBHS, but not in the other two models. The data show that WBHS and ZnSO4 not only prevent TNF-induced mortality but also allow application of TNF in combination with IFN{gamma} as a safe antitumor strategy.


Figure 5
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  		Figure 5. Zinc and WBHS protect against TNF/IFN{gamma} in three different tumor models. C57BL/6 mice were inoculated with three different tumors [i.e., a B16BL6 melanoma (A and B), a PG19 melanoma (C and D), or a Lewis lung carcinoma (E and F)]. In all three cases, tumor-bearing mice were divided randomly into six groups and received PBS, WBHS, or ZnSO4, either alone (n = 8) or in combination with TNF/IFN{gamma} (n = 10). Tumor growth/regression was monitored (A, C, and E) and mortality was recorded until 5 d after the end of the treatment (no further deaths occurred).

 
The protection of ZnSO4 against TNF/IFN{gamma} in tumor-bearing mice also depends on HSP70. We wondered whether the hsp70.1 gene is also critical in the protection against TNF/IFN{gamma} in tumor-bearing mice. B16BL6 tumors were inoculated in C57BL/6 mice (n = 19) and in hsp70.1-deficient mice (n = 22) and the protection of ZnSO4 in the drinking water was evaluated exactly as described above. As can be seen in Fig. 6 , zinc protected significantly in the WT mice (P = 0.037), but not in the hsp70.1 knockout mice (P = 0.157). Hence, zinc protected significantly better in the WT mice compared with the knockout mice (P = 0.004).


Figure 6
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  		Figure 6. Protection of ZnSO4 against TNF/IFN{gamma} lethality in tumor-bearing mice critically depends on HSP70. C57BL/6 (n = 19) and hsp70.1-deficient (n = 22) mice were inoculated with B16BL6 melanoma cells and treated with water or 25 mmol/L ZnSO4 and a therapy with TNF/IFN{gamma}. Survival was recorded up until the end of the treatment (no further deaths occurred).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It has been known for a long time that treatment of tumor-bearing mice with TNF leads to regression of the tumors. This effect is even more pronounced when TNF is combined with IFN{gamma} or with chemotherapeutics (2). The antitumor effect of TNF seems to result from modulation/modification of the integrin {alpha}vß3 specifically in the tumor neovascular endothelial cells (23). The antitumor effect of TNF/IFN{gamma} leads to complete regression of the tumors but is associated with considerable toxicity. Therefore, TNF is currently applied in locoregional treatment schedules only (6, 7). The very powerful proinflammatory nature of TNF is the source of its toxicity. Indeed, TNF is produced by macrophages on stimulation with different agents (e.g., endotoxin; refs. 1, 24). Hence, TNF plays a central mediating role in inflammatory disorders, such as rheumatoid arthritis and IBD (8, 9). In patients suffering from these pathologies, TNF-blocking agents, such as infliximab, have proven to be very effective (25, 26). The major dose-limiting toxicities after TNF injection were found to be liver toxicity, drop in blood pressure (shock), and bowel necrosis (3). TNF is a very strong inducer of gene expression through activation of nuclear factor-{kappa}B (NF-{kappa}B), mitogen-activated protein kinase (MAPK), and other kinases, and it is expected that the toxic response is due to the effects of products of newly expressed genes. Because the antitumor and shock-inducing properties of TNF are based on different pathways (noninflammatory and inflammatory, respectively), we reasoned that inhibition of the inflammation induced by TNF could lead to a safer use of TNF in cancer patients without compromising the antitumor effects.

Zinc is a heavy metal that has been shown to possess clear anti-inflammatory properties. In rat and pig models of endotoxemia, protection by zinc and induction of HSP70 have been described, although no causal relationship between both activities were shown (15, 16). In previous experiments, we found that treatment of mice for 7 days with ZnSO4 in the drinking water increases the expression of ~2,700 genes, of which the inducible hsp70.1 gene is a major and recurrent one.5 Furthermore, we had reported that WBHS treatment of mice protected them from TNF-induced shock in a strictly HSP70-dependent way.

The HSPs form a highly conserved family of stress proteins with very diverse functions. Several genes encoding HSP70 are inducible by many stress factors, including environmental stress, oxidative stress, and inflammation. In mice, the major inducible HSP70-encoding genes are hsp70.1 and hsp70.3, the former being the more strongly induced.

We here investigated whether zinc could protect against inflammatory shock induced by a single bolus injection of TNF. We found that this indeed is the case. Simple addition of ZnSO4 to the drinking water of mice conferred dose-responsive protection against TNF-induced hypothermia, gene induction, and consequent death. The protective effect of zinc on TNF-induced cell death in the bowel is also very interesting because this toxicity has been reported to be strongly associated with the final lethal outcome (4, 5). In TNF-treated animals, apoptosis of epithelial cells leads to loss in architecture of the intestine. This might result in an increased uptake in the circulation of gut-derived endotoxins and in a drastic sensitization to TNF (4). We believe that the inhibition of apoptosis of enterocytes by HSP70 (induced by zinc) prevents such an increased uptake and helps to rescue the organism. Indeed, sterilization of the gut by antibiotics significantly prevents TNF-induced lethality.5 Because other groups have shown that zinc affects the gut flora only very minimally (27, 28), we believe that the effect of zinc on the jejenum is indeed through HSP70, at the level of inhibition of apoptosis, as our data clearly show. It has been shown (29) that zinc inhibits endonucleases that are involved in apoptosis (29) and that zinc blocks caspase-3 activity (30). However, others have shown that caspase inhibition does not protect against TNF in vivo (31). Interestingly, it has been reported that HSP70 inhibits the recruitment of procaspase-9 to the Apaf-1/apoptosome complex, thereby preventing cytochrome c– and ATP-induced apoptosis (5).

We then show that zinc induces HSP70 expression in mice, particularly in the liver and small intestine. This is a more specific expression pattern compared with WBHS, which also induced HSP70 in other organs, such as colon and lung (5). In hsp70.1-deficient mice generated by gene targeting, zinc induced less HSP70 in the small and large intestines and the protective effect of zinc was completely absent. The hsp70.1 knockout mice, which are not overly sensitive to TNF and have no obvious phenotypic changes, therefore show that induction of HSP70 is a necessary step in the protection conferred by zinc. Because we also found that constitutive and ubiquitous expression of HSP70 in transgenic mice leads to protection against TNF, it is tempting to conclude that HSP70 is sufficient to protect against lethal inflammation.

The mechanism by which HSP70 could protect against TNF-induced inflammatory shock remains unclear (and forms the basis of our ongoing research). We believe that the answer lies in the kinetics of gene induction, which are considerably changed by zinc (through HSP70) from persistent to transient. Indeed, zinc seems to inhibit the late phase of gene induction by TNF, as if zinc induces an anti-inflammatory, gene repressing mechanism. Similar findings were reported by Klosterhalfen et al. (15) in a pig model of endotoxemia. TNF-induced gene induction proceeds by activation of major players, such as NF{kappa}B and MAPKs. It has been shown that HSP70 interacts with Bag-1 (32), which under physiologic conditions interacts with Raf-1 (33). The latter interacts with MAPK kinase, leading to activation of transcription factors involved in cell growth and differentiation (34). After induction, HSP70 and Raf-1 compete for Bag-1 binding, resulting in displacement of Bag-1/Raf-1 by Bag-1/HSP70 (34). Hence, HSP70 may affect the activation of transcription factors involved in the induction of IL-6 and NO synthase. Other mechanisms by which HSP70 may inhibit TNF-induced gene induction include enhanced renaturation of I{kappa}B (the major NF-{kappa}B inhibitor), inhibition of the I{kappa}B kinase (IKK) complex, binding to IKK{gamma}, or induction of MAPK phosphatases (34). It remains to be investigated which of the possible mechanisms are at work in this model.

Here, we show the role of ZnSO4 as a strong anti-inflammatory agent in a Th1 cytokine-mediated disease. In addition, asthma, a typical Th2-mediated disease, was recently also linked with zinc. It was shown that nutritional Zn deficiency worsens allergic inflammation. In addition, Zn supplementation led to significant improvement in acute and chronic asthma mouse models (35, 36).

The therapeutic power of the anti-inflammatory zinc is clear from the antitumor experiments. We show that treatment with zinc (or WBHS) can protect the host against the toxic effects of the combination of TNF and IFN{gamma}, without compromising any antitumor activity. The protection conferred by zinc in this anticancer protocol is clearly also depending on HSP70. We believe that these data will form the basis for the development of a safer antitumor treatment based on TNF/IFN{gamma}.


   Acknowledgments
 
Grant support: Interuniversitaire Attractiepolen, Fortis bank assurances, and the onds voor Wetenschappelijk Onderzoek Vlaanderen and Belgische Vereniging Tegen Kanker.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank L. Van Geert, J. De Backer, and M. Goessens for animal care; W. Burms for doing IL-6 bioassays; G. Pagoulatos (University of Ioannina, Ioannina, Greece) for providing the TG mice; and A. Bredan for editing the manuscript.


   Footnotes
 
Note: W. Van Molle and M. Van Roy contributed equally to this work.

5 Unpublished data.. Back

Received 11/27/06. Revised 3/12/07. Accepted 4/20/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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P. J. Smith, M. Wiltshire, E. Furon, J. H. Beattie, and R. J. Errington
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