Cholera Toxin Pretreatment Protects Against Tumor Necrosis Factor Lethality Without Compromising Tumor
Response to Therapy
Mark I. Block, MD; H. Richard Alexander, MD; Jeffrey A. Norton, MD \s=b\ Antitumor therapy with tumor necrosis factor is limited by systemic toxic effects. We studied whether cholera toxin, a bacterial exotoxin that adenosine diphosphate\p=n-\ribosylates the \g=a\-subunitof Gs proteins, could separate the lethal from the antitumor effects of tumor necrosis factor. A single dose of intravenous cholera toxin protected non\p=n-\tumor-bearing
a lethal dose of Escherichia coli endotoxin administered 6 or 24 hours later. On the basis of these results, tumor-bearing mice were randomized to receive either cholera toxin or saline, followed 6 hours later by either human tumor necrosis factor (400 \g=m\g/kg) or saline. Tumor-bearing mice pretreated with cholera toxin had (1) reduced treatment-related mortality (0/11 vs 5/11 for saline controls) and (2) tumor regression similar to that of controls. In a separate experiment in tumor-bearing mice, intravenous human tumor necrosis factor treatment induced an increase in serum levels of murine tumor necrosis factor to a peak of 500 pg/mL at 1 hour in saline-pretreated controls, while a similar increase could not be detected in those mice pretreated with cholera toxin. These results suggest that pretreatment with cholera toxin can reduce the endogenous tumor necrosis factor response to administered tumor necrosis factor and separate the lethal from the antitumor effects. Cholera toxin may prove to be a useful tool for investigating the mechanisms underlying the varied effects of tumor necrosis factor.
mice from
(Arch Surg. 1992;127:1330-1334)
severely limited by its substantial systemic toxic ef¬
fects, manifested primarily as circulatory shock and death.3
The variety of responses to tumor necrosis factor ad¬ ministration in vitro and in vivo has tempted many inves¬ tigators to explore strategies for separating the desirable effects of tumor necrosis factor therapy from the undesir¬ able consequences. While attempts have been made to limit toxic effects, such approaches generally result in con¬ comitant limitation in the therapeutic response4 and pro¬ vide little insight into the mechanisms of action of tumor necrosis factor. Separation of the lethal from the antitumor effects of tumor necrosis factor ultimately depends on an understanding of the mechanisms involved, thereby per¬ mitting selective exploitation or enhancement of the desir¬ able and limitation or avoidance of the undesirable. The use of agents designed to interfere selectively with poten¬ tial pathways of tumor necrosis factor signaling, at the level of either intracellular or intercellular communication, offers the potential to shed light on those mechanisms re¬ sponsible for specific effects of tumor necrosis factor and may therefore lead to improved prospects for antitumor
therapy. A variety of bacterial toxins have been shown to inter¬ fere with normal signal transduction by specifically adenosine
diphosphate-ribosylating the a-subunits of G pro¬
teins, membrane-associated guanosine triphosphate-
binding heterotrimers responsible for coupling a wide variety of receptor-ligand complexes to their respective
necrosis factor is a pluripotent cytokine with cytotoxic activity in vitro and antitumor effects in vivo. It has been identified as an important mediator of septic shock1 and, when administered intravenously, can cause hemorrhagic necrosis and regression of tumors in mice.2
Tumor
Although these experimental findings are encouraging, application of tumor necrosis factor therapy has
clinical
Accepted for publication June 21, 1992. From the Surgical Metabolism Section, Surgery Branch,
been
National
Cancer Institute, National Institutes of Health, Bethesda, Md. Dr Norton is now with Washington University School of Medicine, St Louis, Mo. Presented at the 45th Annual Cancer Symposium of the Society of Surgical Oncology, New York, NY, March 18, 1992. Reprint requests to Washington University School of Medicine, Box 8109, 660 S Euclid Ave, St Louis, MO 63110 (Dr Norton).
effector molecules.57 Cholera toxin, the bacterial exotoxin produced by Vibrio cholera, adenosine diphosphateribosylates the a-subunit of Gs proteins.8 Activated Gs stimulates adenylate cyclase and raises intracellular levels of cyclic adenosine monophosphate. Adenosine diphosphate-ribosylation by cholera toxin blocks hydrol¬ ysis of Gsa-bound guanosine triphosphate to guanosine diphosphate, preventing deactivation and therefore pro¬ longing stimulation of adenylate cyclase.6-7 Cholera toxin thus provides a highly specific mechanism for interfering with normal signal transduction and raising intracellular levels of cyclic adenosine monophosphate. Experimental findings associated with the use of cholera toxin have suggested that this agent may provide insight into the mechanisms of tumor necrosis factor action. Its use in vitro has been shown to block tumor necrosis factor
Downloaded From: http://archsurg.jamanetwork.com/ by a University of Michigan User on 06/17/2015
production by macrophages9
and enhance the cytotoxic effects of tumor necrosis factor in sensitive cell lines (L929).10 Although in vivo use has been limited, prelimi¬ nary data demonstrate that mice pretreated with cholera toxin survive the lethal effects of the Schwartzman reac¬ tion, a tumor necrosis factor-mediated phenomenon, while exhibiting no alteration in serum levels of interleukin 6 compared with control animals.11 These data suggest that cholera toxin may have the ability to interfere selec¬ tively with specific mechanisms of tumor necrosis factor action, without altering others. In this study, we investi¬ gated whether cholera toxin could protect tumor-bearing mice from the lethal effects of tumor necrosis factor admin¬ istration without altering the tumor response, and therefore provide a potential tool for the dissection of the various mechanisms involved in tumor necrosis factor action. MATERIALS AND METHODS Animals Female C57B1/6 mice (19 to 21 g) were group housed in the
animal care facility of the National Cancer Institute of the National Institutes of Health, Bethesda, Md, with access to food and water ad libitum. All experiments were conducted in compliance with the provisions of the Animal Care and Use Committee of the National Institutes of Health.
Reagents Cholera toxin was purchased (Sigma Chemical Co, St Louis, Mo). Recombinant human tumor necrosis factor was obtained and had a specific activity of 1 x 106 to 2.5 x 106 U / mg as measured by the L929 bioassay and an endotoxin level of 30 to 50 pg/6.5xl06 U as measured by the Limulus assay.12 Cholera toxin and human tumor necrosis factor were reconstituted in sterile water and brought to final concentration in normal saline with 0.5% fatty acid-poor endotoxin-free bovine serum albumin (Calbiochem, La folia, Calif). Control animals received an identical volume of saline with 0.5% bovine serum albumin (NS). Endo¬ toxin from Escherichia coli serotype 0127:B8 (LPS, Sigma Chemi¬ cal Co) was reconstituted in saline to a final concentration of 4 mg/mL and administered intraperitoneally.
Determination of Dose and Pretreatment Interval for Cholera Toxin Pilot studies to determine the appropriate dose and pretreat¬ ment interval for administration of cholera toxin were undertaken C57B1/6 female mice weighing 19 to 21 g. in
non-tumor-bearing
Mice were pretreated with either cholera toxin (0.5,2.5,5.0, or 25.0 u,g/kg) or vehicle (NS) via tail vein injection (intravenous). Each of the five groups contained 10 mice. Eighteen hours later, a stan¬ dard lethal dose of E coli endotoxin (40 mg/kg intraperitoneally) was administered and mice were monitored for survival. A sec¬ ond study was then performed to determine the optimal interval for cholera toxin pretreatment with the use of the optimal protec¬ tive dose of cholera toxin. Pretreatment interval was varied from 6 to 96 hours, after which E coli endotoxin (40 mg/kg) was injected intraperitoneally and mice were again monitored for survival. There were 10 animals in each group, except for the 48-hour pre¬ treatment interval group, which contained nine mice.
Assessment of Cholera Toxin Effect on Tumor Necrosis Factor Therapy Mice bearing 10-day subcutaneous tumors were ear-tagged and randomized into four groups: pretreatment with NS or cholera toxin (2.5 u.g/kg) intravenously followed 6 hours later by NS or human tumor necrosis factor (400 u,g/kg) intravenously. Mea¬ surements of tumor size
Measurement of Serum Murine Tumor Necrosis Factor Levels Female C57B1/6 mice weighing 19 to 21 g with 10-day subcu¬ taneous tumors were pretreated with either cholera toxin (2.5 u.g/kg) or NS intravenously followed 24 hours later by human
(400 u.g/kg intravenously). Serum samples collected by retro-orbital puncture immediately before and 1, 2, and 3 hours after human tumor necrosis factor administra¬ tion (n=2, 7, 6, and 7 for cholera toxin-pretreated mice 0,1,2, and 3 hours after human tumor necrosis factor administration, re¬ spectively; n=3, 6, 6, and 4 for NS-pretreated mice 0, 1, 2, and 3 hours after human tumor necrosis factor administration, respec¬ tively). Different mice were used for collection of each serum sample at each time point, and serum levels of murine tumor ne¬ crosis factor (mTNF) were measured by a specific enzyme-linked immunosorbent assay for mTNF (Genzyme Corp, Mass). The assay did not cross-react with human tumor necrosis factor, and the lower limit of detection as stated by the manufac¬ turer was 100 pg/mL of mTNF. tumor necrosis factor
were
Cambridge,
Statistics Survival curves for animals in the pilot studies of cholera toxin dose and pretreatment interval were constructed by the method of Kaplan and Meier and analyzed by the Breslow modification of the Kruskall-Wallis test. Treatment-related mortality in tumor-bearing mice was compared by means of Fisher's Exact Test. Tumor sizes and serum mTNF levels are presented as mean±SEM. Comparisons between tumor sizes and serum mTNF levels at given times were made with a two-tailed unpaired Student's t test. A P value of less than .05 was considered significant.
RESULTS
Experiments in Non-Tumor-Bearing Mice Studies comparing mortality of non-tumor-bearing mice after intraperitoneal administration of £ coli endo¬ toxin demonstrated improved survival in animals pre¬ treated with either 0.5 or 2.5 p-g/kg of cholera toxin com¬ pared with NS-pretreated controls (Fig 1, top; P=.0001 and P
Response to Therapy
Mark I. Block, MD; H. Richard Alexander, MD; Jeffrey A. Norton, MD \s=b\ Antitumor therapy with tumor necrosis factor is limited by systemic toxic effects. We studied whether cholera toxin, a bacterial exotoxin that adenosine diphosphate\p=n-\ribosylates the \g=a\-subunitof Gs proteins, could separate the lethal from the antitumor effects of tumor necrosis factor. A single dose of intravenous cholera toxin protected non\p=n-\tumor-bearing
a lethal dose of Escherichia coli endotoxin administered 6 or 24 hours later. On the basis of these results, tumor-bearing mice were randomized to receive either cholera toxin or saline, followed 6 hours later by either human tumor necrosis factor (400 \g=m\g/kg) or saline. Tumor-bearing mice pretreated with cholera toxin had (1) reduced treatment-related mortality (0/11 vs 5/11 for saline controls) and (2) tumor regression similar to that of controls. In a separate experiment in tumor-bearing mice, intravenous human tumor necrosis factor treatment induced an increase in serum levels of murine tumor necrosis factor to a peak of 500 pg/mL at 1 hour in saline-pretreated controls, while a similar increase could not be detected in those mice pretreated with cholera toxin. These results suggest that pretreatment with cholera toxin can reduce the endogenous tumor necrosis factor response to administered tumor necrosis factor and separate the lethal from the antitumor effects. Cholera toxin may prove to be a useful tool for investigating the mechanisms underlying the varied effects of tumor necrosis factor.
mice from
(Arch Surg. 1992;127:1330-1334)
severely limited by its substantial systemic toxic ef¬
fects, manifested primarily as circulatory shock and death.3
The variety of responses to tumor necrosis factor ad¬ ministration in vitro and in vivo has tempted many inves¬ tigators to explore strategies for separating the desirable effects of tumor necrosis factor therapy from the undesir¬ able consequences. While attempts have been made to limit toxic effects, such approaches generally result in con¬ comitant limitation in the therapeutic response4 and pro¬ vide little insight into the mechanisms of action of tumor necrosis factor. Separation of the lethal from the antitumor effects of tumor necrosis factor ultimately depends on an understanding of the mechanisms involved, thereby per¬ mitting selective exploitation or enhancement of the desir¬ able and limitation or avoidance of the undesirable. The use of agents designed to interfere selectively with poten¬ tial pathways of tumor necrosis factor signaling, at the level of either intracellular or intercellular communication, offers the potential to shed light on those mechanisms re¬ sponsible for specific effects of tumor necrosis factor and may therefore lead to improved prospects for antitumor
therapy. A variety of bacterial toxins have been shown to inter¬ fere with normal signal transduction by specifically adenosine
diphosphate-ribosylating the a-subunits of G pro¬
teins, membrane-associated guanosine triphosphate-
binding heterotrimers responsible for coupling a wide variety of receptor-ligand complexes to their respective
necrosis factor is a pluripotent cytokine with cytotoxic activity in vitro and antitumor effects in vivo. It has been identified as an important mediator of septic shock1 and, when administered intravenously, can cause hemorrhagic necrosis and regression of tumors in mice.2
Tumor
Although these experimental findings are encouraging, application of tumor necrosis factor therapy has
clinical
Accepted for publication June 21, 1992. From the Surgical Metabolism Section, Surgery Branch,
been
National
Cancer Institute, National Institutes of Health, Bethesda, Md. Dr Norton is now with Washington University School of Medicine, St Louis, Mo. Presented at the 45th Annual Cancer Symposium of the Society of Surgical Oncology, New York, NY, March 18, 1992. Reprint requests to Washington University School of Medicine, Box 8109, 660 S Euclid Ave, St Louis, MO 63110 (Dr Norton).
effector molecules.57 Cholera toxin, the bacterial exotoxin produced by Vibrio cholera, adenosine diphosphateribosylates the a-subunit of Gs proteins.8 Activated Gs stimulates adenylate cyclase and raises intracellular levels of cyclic adenosine monophosphate. Adenosine diphosphate-ribosylation by cholera toxin blocks hydrol¬ ysis of Gsa-bound guanosine triphosphate to guanosine diphosphate, preventing deactivation and therefore pro¬ longing stimulation of adenylate cyclase.6-7 Cholera toxin thus provides a highly specific mechanism for interfering with normal signal transduction and raising intracellular levels of cyclic adenosine monophosphate. Experimental findings associated with the use of cholera toxin have suggested that this agent may provide insight into the mechanisms of tumor necrosis factor action. Its use in vitro has been shown to block tumor necrosis factor
Downloaded From: http://archsurg.jamanetwork.com/ by a University of Michigan User on 06/17/2015
production by macrophages9
and enhance the cytotoxic effects of tumor necrosis factor in sensitive cell lines (L929).10 Although in vivo use has been limited, prelimi¬ nary data demonstrate that mice pretreated with cholera toxin survive the lethal effects of the Schwartzman reac¬ tion, a tumor necrosis factor-mediated phenomenon, while exhibiting no alteration in serum levels of interleukin 6 compared with control animals.11 These data suggest that cholera toxin may have the ability to interfere selec¬ tively with specific mechanisms of tumor necrosis factor action, without altering others. In this study, we investi¬ gated whether cholera toxin could protect tumor-bearing mice from the lethal effects of tumor necrosis factor admin¬ istration without altering the tumor response, and therefore provide a potential tool for the dissection of the various mechanisms involved in tumor necrosis factor action. MATERIALS AND METHODS Animals Female C57B1/6 mice (19 to 21 g) were group housed in the
animal care facility of the National Cancer Institute of the National Institutes of Health, Bethesda, Md, with access to food and water ad libitum. All experiments were conducted in compliance with the provisions of the Animal Care and Use Committee of the National Institutes of Health.
Reagents Cholera toxin was purchased (Sigma Chemical Co, St Louis, Mo). Recombinant human tumor necrosis factor was obtained and had a specific activity of 1 x 106 to 2.5 x 106 U / mg as measured by the L929 bioassay and an endotoxin level of 30 to 50 pg/6.5xl06 U as measured by the Limulus assay.12 Cholera toxin and human tumor necrosis factor were reconstituted in sterile water and brought to final concentration in normal saline with 0.5% fatty acid-poor endotoxin-free bovine serum albumin (Calbiochem, La folia, Calif). Control animals received an identical volume of saline with 0.5% bovine serum albumin (NS). Endo¬ toxin from Escherichia coli serotype 0127:B8 (LPS, Sigma Chemi¬ cal Co) was reconstituted in saline to a final concentration of 4 mg/mL and administered intraperitoneally.
Determination of Dose and Pretreatment Interval for Cholera Toxin Pilot studies to determine the appropriate dose and pretreat¬ ment interval for administration of cholera toxin were undertaken C57B1/6 female mice weighing 19 to 21 g. in
non-tumor-bearing
Mice were pretreated with either cholera toxin (0.5,2.5,5.0, or 25.0 u,g/kg) or vehicle (NS) via tail vein injection (intravenous). Each of the five groups contained 10 mice. Eighteen hours later, a stan¬ dard lethal dose of E coli endotoxin (40 mg/kg intraperitoneally) was administered and mice were monitored for survival. A sec¬ ond study was then performed to determine the optimal interval for cholera toxin pretreatment with the use of the optimal protec¬ tive dose of cholera toxin. Pretreatment interval was varied from 6 to 96 hours, after which E coli endotoxin (40 mg/kg) was injected intraperitoneally and mice were again monitored for survival. There were 10 animals in each group, except for the 48-hour pre¬ treatment interval group, which contained nine mice.
Assessment of Cholera Toxin Effect on Tumor Necrosis Factor Therapy Mice bearing 10-day subcutaneous tumors were ear-tagged and randomized into four groups: pretreatment with NS or cholera toxin (2.5 u.g/kg) intravenously followed 6 hours later by NS or human tumor necrosis factor (400 u,g/kg) intravenously. Mea¬ surements of tumor size
Measurement of Serum Murine Tumor Necrosis Factor Levels Female C57B1/6 mice weighing 19 to 21 g with 10-day subcu¬ taneous tumors were pretreated with either cholera toxin (2.5 u.g/kg) or NS intravenously followed 24 hours later by human
(400 u.g/kg intravenously). Serum samples collected by retro-orbital puncture immediately before and 1, 2, and 3 hours after human tumor necrosis factor administra¬ tion (n=2, 7, 6, and 7 for cholera toxin-pretreated mice 0,1,2, and 3 hours after human tumor necrosis factor administration, re¬ spectively; n=3, 6, 6, and 4 for NS-pretreated mice 0, 1, 2, and 3 hours after human tumor necrosis factor administration, respec¬ tively). Different mice were used for collection of each serum sample at each time point, and serum levels of murine tumor ne¬ crosis factor (mTNF) were measured by a specific enzyme-linked immunosorbent assay for mTNF (Genzyme Corp, Mass). The assay did not cross-react with human tumor necrosis factor, and the lower limit of detection as stated by the manufac¬ turer was 100 pg/mL of mTNF. tumor necrosis factor
were
Cambridge,
Statistics Survival curves for animals in the pilot studies of cholera toxin dose and pretreatment interval were constructed by the method of Kaplan and Meier and analyzed by the Breslow modification of the Kruskall-Wallis test. Treatment-related mortality in tumor-bearing mice was compared by means of Fisher's Exact Test. Tumor sizes and serum mTNF levels are presented as mean±SEM. Comparisons between tumor sizes and serum mTNF levels at given times were made with a two-tailed unpaired Student's t test. A P value of less than .05 was considered significant.
RESULTS
Experiments in Non-Tumor-Bearing Mice Studies comparing mortality of non-tumor-bearing mice after intraperitoneal administration of £ coli endo¬ toxin demonstrated improved survival in animals pre¬ treated with either 0.5 or 2.5 p-g/kg of cholera toxin com¬ pared with NS-pretreated controls (Fig 1, top; P=.0001 and P
