De-repression of Anti-Cancer Immunity by N-Acetylcysteine
Author: Emiliano Rapoport
Abstract
This paper describes a method of treating cancer through de-repression of anticancer immune responses using a drug that is already in clinical use for treatment of acetaminophen overdose.
Summary
§ We propose a small pilot study administering N-acetylcysteine to advanced cancer patients with the objectives of:
o Suppressing neutrophil hyperactivation
o Suppressing markers of oxidative stress/inflammation
o Restoring TCR-zeta chain expression and IFN-g production in patient T cells
Why?
§ Tumor patients suffer from antigen-specific and non-specific suppression of T cell function. This is why current immunotherapies are failing. If the T cells don’t work how will cancer vaccines do anything?
§ Suppression of T cell function is due in part to H2O2 produced by activated neutrophils that are circulating systemically.
§ Co-culture of neutrophils from cancer patients with healthy person T cells causes T cells to lose zeta-chain of TCR. This results in functional inactivation.
§ Addition of catalase to cancer patient neutrophils blocks ability to suppress healthy T cells.
§ Systemic neutrophil activation in cancer patients can be easily assessed: The neutrophils in cancer patients co-purify with lymphocytes on Ficoll gradient because of their activation status. Flow cytometry of Ficoll gradient separated cells shows distinct differences cells of cancer patients and control patients.
§ N-acetylcysteine is a safe drug clinically used for treatment of acetominophen poisoning and lung disorders.
§ N-acetylcysteine is a promising therapy for blocking neutrophil hyperactivation in cancer patients since:
o It blocks neutrophil hyperactivation in sepsis patients.
o It blocks neutrophil hyperactivation clinically in inflammatory conditions.
o Restores immune function in animal models of sepsis-induced immune suppression.
o Blocks angiogenesis
o Directly suppresses proliferation of cancer cells but not normal counterparts in vitro.
Is there such a thing as cancer-associated immune suppression?
Immune suppression by cancer has been well-documented in advanced cancer patients possessing a variety of malignancies. These include pancreatic cancer, breast cancer, renal cancer, colorectal cancer and melanoma. Suppression is noted by diminished T cell proliferative response, diminished ability to produce IFN-g, and diminished ability to induce recall responses to normal antigens [1-8].
What is the importance of tumor-induced immune suppression?
Immune suppression does not allow for proper eradication of tumors by immunotherapy, or by the body’s natural mechanisms. Correlation between immune suppression and poor prognosis has been extensively noted [9-11].
What is the molecular basis for tumor-induced immune suppression?
The tumor cells induce cleavage of the T cell receptor zeta (TCR-z) chain through a caspase-3 dependent manner [12, 13]. This is both FasL-dependent and independent. Since TCR-z is critical for signal transduction, the T cells become unable to respond to tumor antigens. Originally, the suppressed level of TCR-z was described in tumor bearing mice [14, 15] and subsequently in patients. Olivera Finn from University of Pittsburgh described suppressed TCR-z expression in advanced cancer patients [16]. Similar data has been reported for a wide variety of cancers [17-21]. The correlation between suppressed TCR-z and suppressed IFN-g production has also been reported [17].
How does the tumor suppress TCR-z?
The cause of TCR-z suppression has been attributed, at least in part, to reactive oxygen radicals produced by:
A) The inflammatory activity occurring inside the tumor (it is well established that there is a constant area of necrosis intratumorally
B) Macrophages associated with the tumor.
C) Neutrophils activated directly by the tumor, or by the tumor associated macrophages.
Tumors usually associated with macrophage infiltration, this is correlated with tumor stage and is believed to contribute to tumor progression by stimulation of angiogenesis [22-24]. Cytokines such as M-CSF [22] and VEGF [25] produced by tumor infiltrating macrophages are essential for tumor progression to malignancy. In fact, tumors implanted into M-CSF deficient op/op mice (they lack macrophages) do not metastasize or become vascularized [26]. Tumor-associated macrophages possess an activated phenotype and release various inflammatory mediators such as cyclo-oxygenase metabolites [27, 28], TNF-a [29], and IL-6 [30].
In addition, tumor associated macrophages produce large amounts of free radicals such as NO, OH, and H2O2 [31-33]. The high levels of macrophage activation in cancer patients is illustrated by high serum levels of neopterin, a feature that is associated with poor prognosis [34].
What evidence is there that the tumor itself causes non-specific inflammation that could activate neutrophils?
In addition to oxidative stress elaborated by tumor associated macrophages, the presence of the tumor itself causes systemic changes associated with chronic inflammation. Erythrocyte sedimentation ration, C-reactive protein and IL-6 are markers of inflammatory stress used to designate progression of diseases such as arthritis [35, 36]. Interestingly advanced cancer patients possess all of these inflammatory markers [37-41]. Another marker of chronic inflammation is decreased albumin synthesis by the liver, this is also seen in cancer patients and is believed to contribute, in part, to cachexia [42, 43]. In addition, the inflammatory marker fibrinogen D-dimers is also higher in cancer patients as opposed to controls [44-46].
How can we assess activated neutrophils in cancer patients?
Schmielau et al reported that in patients with a variety of cancers, activated neutrophils are circulating in large numbers [16]. These neutrophils secrete reactive oxygen radicals such as hydrogen peroxide which trigger suppression of TCR-z and IFN-g production. This was demonstrated by co-incubation of the neutrophils from cancer patients with lymphocytes from healthy volunteer. A profound suppression of TCR-z expression was seen. Evidence for the critical role of hydrogen peroxide was shown by the fact that addition of catalase suppressed TCR-z downregulation. A simple method of assessing the number of circulating activated neutrophils was described in the same paper. This method involves collecting peripheral blood from patients, spinning the blood on a density gradient such as Ficoll, and collecting the lymphocyte fraction. While in healthy volunteers the lymphocyte fraction contained primarily lymphocytes, in cancer patients the lymphocyte fraction contained both lymphocytes and a large number of neutrophils. The reason why these neutrophils are present in the lymphocyte fraction is because activation alters their density so that they co-purify differently on the gradient.
Any evidence for clinical improvement after neutrophil depletion?
A potential indication of the importance of activated neutrophils to cancer progression is provided by Tabuchi et al who show that removal of granulocytes from the peripheral blood of cancer patients resulted in reduced tumor size, unfortunately, the study was performed in only 2 patients [47].
What is known about oxidative stress and immune response?
As a mechanism to compensate for immune over-activation, mediators of inflammation have immune suppressive properties. This is best illustrated in the immune suppression seen following immune hyperactivation such as in septic shock. Following the primary scepticemia, patients are systemically immune compromised due to circulating immune suppressive factors that are released in response to the inflammatory stress. This suppression is termed compensatory anti-inflammatory response syndrome (CARS) and is associated with many opportunistic infections and deactivation [48]. The clinical importance of CARS immune suppression is seen in that sepsis survivors show normal T-cell proliferation and IL-2 release, whereas those that succumb possess suppressed T cell responses [49].
What do CARS and cancer have in common?
Interestingly immune suppressive mediators associated with CARS such as PGE2, TGF-b, and IL-10 are also associated with cancer-induced immune suppression [50]. The role of oxidative stress in sepsis-induced immune suppression was recently demonstrated in experiments where administration of antioxidants (ascorbic acid or n-acetylcysteine) to animals undergoing experimental sepsis blocked immune suppression [51]. Another example of the potential for antioxidants to stimulate immune response in an inflammatory condition is in patients with Duke’s C and D colorectal cancer who were administred of a daily dose of 750 mg of vitamin E for 2 weeks. This resulted in restoration of IFN-g and IL-2 production [52].
Has N-acetylcysteine been used in the past besides acetominophen poisoning?
Yes, to name a few…
A) The problem of uncontrolled inflammation is seen in sepsis. Although as a monotherapy n-acetylcysteine has little clinical effect, therapeutic administration of n-acetylcysteine results in suppression of the constitutively activated neutrophils seen in these patients [53].
B) Administration of n-acetylcysteine to smokers results in suppression of markers of oxidative stress [54].
C) Oral n-acetylcysteine blocks angiogenesis and suppresses growth of Kaposi Sarcoma [55].
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