Article by: Dana F. Flavin, Dr. med univ, Innsbruck, Austria
Director, Foundation for Collaborative Medicine and Research, USA
www.collmed.org

Direct suppression/inactivation of oxygen radicals:

Oxygen radicals from various different oxidases, including xanthine oxidase (XO) and NADPH oxidase (NADPHO) in macrophages and lymphocytes, as well as the circulation in the serum of XO, are responsible for the production of toxic substances such as peroxynitrite, OH radicals, hydrogen peroxide, etc. which are toxic to foreign proteins, bacteria, viruses, and cancer cells. This ability to produce large amounts of oxygen radicals is not seen in cancer cells themselves, but also in the cancer patients’ immune cells. They are weakened against cancer cell attack, partially because of the nitric oxide (NO) released by tumor cells.   Excellent work by Prof de Groot of Essen, indicated by adding exogenous xanthine oxidase ( XO) in hepatoma cells, hydrogen peroxide was produced to destroy the hepatoma cells. However, since XO is inhibited in cancer patients from NO generated by tumor cells and most likely other factors not yet known, the production of hydrogen peroxide that would normally destroy a cancer cell is not produced. NO from eNOS in cancer cells can travel through membranes and over long distances in the body. It has even been shown that cGMP is stimulated in the liver in colon cancer patients and this is most likely from growth factors and NO released from the tumor cells.  NO also is co linked to VEGF which in turn increases the antiapoptotic gene bcl-2. The other important influence of NO is in its inhibition of the proapoptoic caspases cascade. This in turn protects the cells from intracellular preprogrammed death.

One other important alteration by nitric oxide in immune suppression in relation to oxygen radicals is its inhibitory effect on the binding of leukocytes (PMN) at the endothelial surface. This is normally an early stage for toxicity against bacteria, or “foreign” substances. However, this inability of binding alters this form of toxicity too.

Inhibition of inducible Nitric Oxide Synthase (iNOS): In macrophages, leukocytes and T-killer cells, the production of a fast generated NO from iNOS causes a very fast reaction with superoxide radicals from oxidases. It is enhanced by free iron, and is responsible for the toxicity from these cells. One of these toxic products generated is peroxynitrite. When the iNOS is down regulated or inhibited, or the oxygen radicals totally suppressed, there is a tremendous reduction in toxicity to almost null in some cases.

Binding of iron also affects this toxicity. NO from the tumor cells actually suppresses the iNOS, and in addition it reduces oxygen radicals to stop the formation of peroxynitrite in these cells. But NO is not the only inhibitor of iNOS in cancer.

Spermine and spermidine, from the rate limiting enzyme for DNA synthases, ODC, also inhibit iNOS. Since ODC is permanently elevated in these cells without any down regulation, the products are continuously generated.  It is not known why the ODC remains elevated, the enzymes responsible for ODC’s down regulation appear to be altered in cancer cells.

Complex antibodies in cancer patients result in a tolerance in the immune system that decreases the immune response to antigens on the tumors.  This immune suppression can be overcome with plasmapheresis.  Additionally, Freund’s adjuvant reversed the immune suppression to tumor antigens in patients.

Phosphorylated proteins in tumor cells also play an important role in suppression of toxicity to these cancer cells. There is an increase in kinases in these cells which phosphorylate serine, and tyrosine.  The latter is responsible for activation of many growth factors and enzymes. In addition these phosphorylated amino acids suppress iNOS activity, inhibiting toxicity against them from macrophages, leukocytes and lymphocytes.

Another very important kinase is pyruvate dehydrogenase kinase which phosphorylates Pyruvate dehydrogenase blocking the pyruvate from entering the mitochondria and increasing lactic acid formation in the tumors, also then reducing citrate in the cells imperative for Acetyl-CoA in the Krebs cycle.  These are part of the problem in anaerobic glycolysis.  One of the other major kinases is Hexokinase II which binds onto the voltage dependant anion channel (VDAC). This enzyme feeds the cancer cells. When this enzyme is blocked the tumors do not survive, however some tumors do not respond.  HKII’s major substrate 2 deoxyglucose was shown by Pedersen to be important for developing the PET scan.

 

Hexokinase I, on the other hand is important for apoptosis II.

One of the Prostaglandins, Prostaglandin E2, released from tumor cells is also an inhibitor of iNOS, as well as suppressing the immune system.  One begins to see the complicated interactions of tumor substances in suppression toxicity.

Decrease in Lymphocyte Toxicity/Replication:

The lymphocyte immune system of defense against cancer stems from the Th-1 subset of T-cells. These cells are responsible for anti-viral and anti-cancer activities, via their cytokine production including Interleukin-2, (IL-2), and Interleukin-12 which stimulates T-killer cell replication and further activation and release of tumor fighting cytokines. The Th-2 subset of lymphocytes, on the other hand are activated in allergies and parasitic infections to release Interleukin-4 and Interleukin-10. These have respectively inhibitory effects on iNOS and lymphocyte Th-1 activation, but stimulate eosinophils and mast cells, bradykinin release etc.  Mast cells contain histamine which when released increases the T suppressor cells, to lower the immune system and also acts directly on many tumor Histamine receptors to stimulate tumor growth.

Tumor cells release IL-10, and this is thought to be one of the important areas of Th-1 suppression in cancer patients. Interesting enough, IL-10 is also increased in cancer causing viral diseases such as HIV, HBV, HCV, and EBV. This interesting correlation of cancer producing viruses and cancer growth has yet to be further investigated. IL-10 is also a central regulator of cyclooxygenase-2 expression and prostaglandin production in tumor cells stimulating their angiogenesis and NO production. The viruses that can cause cancer, such as HIV and HBV/HCV, cannot be grown in normal cells. Instead, transformed cells are necessary for viral replication. Transformed cancer cells do not produce oxygen radicals therefore the HIV does not lyse the transformed T-cells. HBV/HCV grown in transformed liver cells does not destroy the hepatoma cells. In normal cells the generation of oxygen radicals would cause cell death, and in addition, the nitric oxide in tumor cells even prevents the activation of caspases responsible for apoptosis (programmed cell death).

The main difference in tumor cells vs. Tumor promotion is that in the early stages of carcinogenesis, which we call tumor promotion, one needs a strong immune system, and fewer oxygen radicals to prevent mutations but still enough to destroy the tumor cells should they develop. In later stages of cancer development, the oxygen radicals are decreased around the tumors and in the tumor cells themselves, and the entire cancer fighting Th-1 cell replication and movement are suppressed. The results are a decrease in direct toxicity and apoptosis, which is prevented by NO, a suppression of the macrophage and leukocyte toxicity and finally, a suppression of the T-cell induced tumor toxicity.

Recent data indicates a role of NO in T-cell suppression via two important factors. One is the increase in IL-12p40, and is a decrease in IL-12p35 from NO. The IL-12p40 inhibits T-cell replication. Another factor is that the increase in cGMP in the lymphocytes also decreases their cell replication. Since cGMP is increased by NO, and the NO from tumor cells can travel quite far through membranes and tissues to other organs, including lymph glands, it is not so surprising that the T-cells are suppressed.

 

One last point to emphasize in the effects of NO in cancer is its ability to increase platelet-tumor cell aggregates, which enhances metastases.  It allows the cell to travel more efficiently through the bloods and find new areas to replicate.  The greater the NO production in many types of tumors, the greater the malignancies and the greater the metastatic potential of these tumors.  This is not in all tumor types, but indeed in many, particularly gynecological.

Another factor in Cancer patients is the elevated lactic acid which neutralizes the toxicity and activity of Lymphocyte immune response and mobility.  This lactic acid and hydrogen are released from the tumor cells via the mitochondria.  The lactic acid is also feeding fungi around tumors and that leads to elevated histamine which increases T-suppressor cells.  Histamine alone stimulates many tumor cells. .

T-regulatory cells (formerly,T suppressor cells) down regulate the activity of Natural killer cells and last but not least, the Lactic acid from tumor cells and acidic diets shifts the lymphocyte activity to reduce its efficacy against cancer cells and pathogens in addition to altering the bacteria of the intestinal tract.  The intestinal tract bacteria in cancer cells release sterols that suppress the immune system and down regulate anticancer activity from lymphocytes.

In addition to the lactic acid, adenosine is also released from tumors. Through IL-10, adenosine and other molecules secreted by regulatory T cells, the CD8+ cells can be inactivated to an anergic state. Adenosine up regulates the PD1 receptor in T-1 Lymphocytes and inhibits their activity. The resistance of many human cancers to immune-based therapies, including adoptive immunotherapy and the administration of therapeutic cancer vaccines, has been attributed to tumor-associated immune suppression, due in part to immunosuppressive molecules located within the tumor microenvironment.

Adenosine is a purine nucleoside found within the interstitial fluid of solid tumors at concentrations that are able to inhibit cell-mediated immune responses to tumor cells. It is well established that extracellular adenosine inhibits T lymphocyte activation and effector function, including T cell adhesion to tumor cells and cytotoxic activity, by signaling primarily through A2a and A3 adenosine receptors on the surface of T cells. Importantly, A2a adenosine receptor-deficient mice exhibit enhanced anti-tumor immune responses by CD8+ T cells, as well as a dramatic reduction in the growth of experimental tumors in comparison to wild-type controls.

A2a adenosine receptor signaling has also been implicated in adenosine-mediated inhibition of cytokine production and cytotoxic activity by activated natural killer (NK) cells, although the process of NK cell granule exocytosis is apparently suppressed via a distinct and as yet uncharacterized adenosine novel extracellular receptor.  Adenosine appears to up-regulate the PD1 receptor in T-1 Lymphocytes and inhibits the immune system further.  It is thought by some that adenosine actually binds on the PD-1 receptor, although this is not confirmed.

Mast cells from various types of subgroups have also been proven to influence the immune system by releasing IL-9 which lowers the immune system as well as other cytokines that influence cancer growth.  In addition, the Mast cells with their release of histamine lower the immune system and also stimulate tumor growth and activate the metalloproteinases involved in angiogenesis and metastases.  Lung, thyroid, colon and pancreas cancer are known be greatly influenced by Mast cells and perhaps we shall see this influence in other types of cancers. Mast cells also release heparinase which splits off a VEGF from heparin sulfate allowing angiogenesis.

Most malignant cell lines and experimental tumors release the histamine synthesizing enzyme, L-histidine decarboxylase, and contain high concentrations of endogenous histamine.  Histamine regulates diverse biological responses related to tumor growth including angiogenesis, cell invasion, migration, differentiation, apoptosis and modulation of the immune response.

Myeloid Derived suppressor cells are increased around tumors that are resistant to therapies and they enhance the growth of the tumor cells.  These cells are actually suppressing the immune system and can be down regulated with COX 2 inhibitors or all trans-retinoic acid.  Cimetidine, an antihistamine has been actually shown to increase in apoptosis in MDSC via a separate mechanism than the antihistamine effect. The mechanism appears to be a possible induction of Fas and FasL expression on the MDSC surface and sequentially regulating the caspase dependent apoptosis pathway.

Fibroblasts and tumor development: Cancer-associated fibroblasts (CAFs) form the most preponderant cell type in the solid tumor microenvironment. Given their pervasive role in facilitating tumor growth and metastatic dissemination by activation of VEGF, IL1a, COX2 and other factors, CAFs have emerged as attractive therapeutic targets in the tumor microenvironment. Cancer-associated stromal fibroblasts (CAFs) are the main cellular constituents of reactive stroma in primary and metastatic cancer.  The phenotypical characteristics of CAFs from human colorectal liver metastases (CLMs) and their role in inflammation and cancer progression were analyzed. CAFs displayed a vimentin(+), alpha-smooth-muscle actin(+), and Thy-1(+) phenotype similar to resident portal-located liver fibroblasts (LFs). It was demonstrated that CLMs are inflammatory sites showing stromal expression of interleukin-8 (IL-8), a chemokine related to invasion and angiogenesis. In vitro analyses revealed a striking induction of IL-8 expression in CAFs and LFs by tumor necrosis factor-alpha (TNF-alpha). The effect of TNF-alpha on CAFs is inhibited by the nuclear factor-kappaB inhibitor parthenolide. Conditioned medium of CAFs and LFs similarly stimulated the migration of DLD-1, Colo-678, HuH7 carcinoma cells, and human umbilical vein endothelial cells in vitro. Pretreatment of CAFs with TNF-alpha increased the chemotaxis of Colo-678 colon carcinoma cells by conditioned medium of CAFs; however, blockage of IL-8 activity showed no inhibitory effect. In conclusion, these data raise the possibility that the majority of CAFs in CLM originate from resident LFs. TNF-alpha-induced up-regulation of IL-8 via nuclear factor-kappaB in CAFs is an inflammatory pathway, potentially permissive for cancer invasion that may represent a novel therapeutic target

A-N-acetylgalactosaminidase or Nagalase is a glycoside hydrolase from bacteria and animals and promotes immune suppression by inactivating macrophages.

Alpha-N-acetyl galactosaminidase (alpha-NaGalase) has been reported to accumulate in serum of cancer patients and be responsible for deglycosylation of Gc protein, which is a precursor of Gc-MAF-mediated macrophage activation cascade, finally leading to immunosuppression in advanced cancer patients. It was supposed as one of the mechanisms for immuno-suppression in cancer patients that macrophage activating factor (Gc MAF) could not be produced from a precursor, serum vitamin D-binding protein (Gc protein) due to deglycosylation by a-N-acetyl galactosaminidase (α-NaGalase). It was reported that α-NaGalase increased in cancer patients’ serum (Yamamoto, N. et al., 1998). To develop an immuno modulator of cancer therapy, the mechanism of immuno-suppression was defined by characterization of α-NaGalase in various tumor cells and α-NaGalase inhibitors was designed and synthesized as immuno potentiator.

1) α-NaGalase activities in tumor cell lysates from Hep G2 and HCT116 cells and normal cell lysates from Chang liver cell and isolated rat hepatocytes were measured. A high specific activity of a-NaGalase was found in tumor cell lines compare to normal cells. Because α-NaGalase deglycosylated exo-type substrate specifically, it was necessary to reinvestigate the deactivation mechanism of GcMAF by a-NaGalase.

2) Azasugar derivatives introduced spィイD12ィエD1 carbon to control a torsional angle between hydroxyl groups were designed and synthesized as a α-NaGalase inhibitor and an immunopotentiator, because a sugar-shaped alkaloid, swainsonine, was a α-mannosidase inhibitor and an immunopotentiator. Activities of α-NaGalase inhibition and macrophage activation are now under investigation.

Transforming Growth Factor beta:  TGFb is also increased in cancer patients and suppresses the immune system.  TGF-β inhibits the proliferation of T cells as well as cytokine production via Foxp3-dependent and independent mechanisms. On the one hand, little is known about molecular mechanisms involved in immune suppression via TGF-β; however, recent studies suggest that Smad2 as well as Smad3 play essential roles in Foxp3 induction and cytokine suppression, whereas Th17 differentiation is promoted via the Smad-independent pathway. Mutual suppression of signaling between TGF-β and inflammatory cytokines has been shown to be necessary for the balance of immunity and tolerance.

Cancer-associated stromal fibroblasts (CAFs) are the main cellular constituents of reactive stroma in primary and metastatic cancer.  The phenotypical characteristics of CAFs from human colorectal liver metastases (CLMs) and their role in inflammation and cancer progression were analyzed. CAFs displayed a vimentin(+), alpha-smooth-muscle actin(+), and Thy-1(+) phenotype similar to resident portal-located liver fibroblasts (LFs). It was demonstrated that CLMs are inflammatory sites showing stromal expression of interleukin-8 (IL-8), a chemokine related to invasion and angiogenesis. In vitro analyses revealed a striking induction of IL-8 expression in CAFs and LFs by tumor necrosis factor-alpha (TNF-alpha). The effect of TNF-alpha on CAFs is inhibited by the nuclear factor-kappaB inhibitor parthenolide. Conditioned medium of CAFs and LFs similarly stimulated the migration of DLD-1, Colo-678, HuH7 carcinoma cells, and human umbilical vein endothelial cells in vitro. Pretreatment of CAFs with TNF-alpha increased the chemotaxis of Colo-678 colon carcinoma cells by conditioned medium of CAFs; however, blockage of IL-8 activity showed no inhibitory effect. In conclusion, these data raise the possibility that the majority of CAFs in CLM originate from resident LFs. TNF-alpha-induced up-regulation of IL-8 via nuclear factor-kappaB in CAFs is an inflammatory pathway, potentially permissive for cancer invasion that may represent a novel therapeutic target

Macrophage:  Tumor cells build lactic acid from the change in the metabolism from oxidation to glycolysis, allowing a low oxygen tension in tumor cells.  The hyperpolarized mitochondria does not take in the pyruvate, as the Pyruvate dehydrogenase enzyme is blocked by another enzyme, called Pyruvate dehydrogenase Kinase I.  The pyruvate then goes into lactic acid and shifts the pH in the tumor cells. This acid changes many parameters in the tumor cells and draws in macrophages.  Instead of the macrophages being destructive to the tumor, they shift from an M1 non stimulating macrophage, to an M2 tumor stimulating macrophage releasing growth factors and nitric oxide for angiogenesis and tumor growth. These macrophages in the tumors of the M2 inhibit response to radiation and immunotherapy.    The macrophage factors released pull in the mast cells and they release more immune suppressive factors and stimulate tumor growth.