Re-educating the Immune System

An increasingly apparent reality is that tumour cells release many cytokines and other molecules for the express purpose of converting the immune system from a tumour-suppressing to a tumour-promoting force. “Re-educating” the immune system refers to the task of reversing this tumour-induced state of immune-suppression, allowing the anti-cancer arm of the immune system to function as it should.

When reading the literature on cancer and the immune system, it is easy to be confused by the complexity of terminology for the various immune cells, cytokines, cell surface antigens etc. I will list a few basic points to remember.

Immune system components which destroy or help to destroy cancer cells include:

Cells of the innate immune system

  • Natural killer (NK) cells. Like cytotoxic T-lymphocytes (see below), natural killer cells kill invading cells by releasing cytotoxins such as perforins and granzymes.
  • M1-polarized macrophages release cytokines such as interleukin-12 (IL-12) which promotes a T-helper 1 type immune response, and tumour necrosis factor alpha (TNF-alpha). M1 macrophages can also present antigens to the adaptive immune system (due to the presence of major histocompatibility complex II, or MHC II) and costimulatory molecules CD80 and CD86.

Cells of the adaptive (acquired) immune system

  • Mature dendritic cells, the professional antigen-presenting cells of the immune system. Dendritic cells process and present antigens to T-cells, display the cell surface molecules CD80 and CD86, which stimulate T-cell activation, and major histocompatibility complex II (MHC-II) which is necessary for antigen presentation. Dendritic cells also secrete interleukin-12, stimulating a type 1 immune response.
  • Type 1 T helper cells (Th1). CD4+ cells which secrete cytokines such as interferon-gamma. Th1 cells promote the cell killing abilities of macrophages and CD8+ cytotoxic T lymphocytes.
  • Cytotoxic T Lymphocytes (CTLs), which are recognized as CD8+. These cells can kill cancer cells by releasing various cytotoxins such as perforin and granzymes.

In contrast with the above, immune system components which suppress the cytolytic, anti-cancer arm of the immune system include:

  • M2-polarized macrophages, tumour-associated macrophages and microglia (TAMs), release immunosuppressive cytokines such as interleukin 10.
  • myeloid-derived suppressor cells (MDSC) which release immunosuppressive cytokines such as interleukin-10 and transforming growth factor-beta (TGF-beta). MDSC also release arginase 1, which inhibits T-cell proliferation and induces T-cell apoptosis.
  • T-regulatory cells (Tregs). Tregs are involved in suppressing the active arm of the immune system and play a large role in the immunosuppressive environment found in tumours. Tregs are identified as CD4+ CD25+ Foxp3+.

Clinically validated

Polysaccharide Krestin (PSK)

Isolated from the mushroom known as Turkey tail (Trametes versicolor or Coriolus versicolor). Approved in Japan and clinically validated as an adjuvant to chemotherapy in gastric cancer, colorectal cancer, lung cancer, etc [42].

PDE5 inhibitors: sildenafil (Viagra, Revatio), vardenafil, tadalafil

Sildenafil, a PDE5 inhbitor approved by the FDA in 1998 for male erectile dysfunction, is now being investigated for two potential cancer uses, one of which has made it to a clinical glioma trial. This trial for recurrent high grade glioma is adding sildenafil to sorafenib and valproic acid therapy for the purpose of increasing the passage of sorafenib through the blood-brain barrier. This function of sildenafil, the increasing of blood-tumour barrier permeability (by two different mechanisms) has been validated in a rat model of glioma, where it increased survival in rats when added to chemotherapy [17].

Tadalafil – clinical evidence

Another drug, tadalafil (Cialis, Adcirca), was the third PDE5 inhibitor approved by the FDA for erectile dysfunction, in 2003. Further proof for the immune-stimulating effects of this class of drugs comes in the form of a randomized trial of tadalafil in pre-operative head and neck cancer patients [40, 41]. The results of this trial were published online [40] in the journal Clinical Cancer Research in October 2014. Patients in this study were randomized into one of three groups (3:3:1 ratio): 10 mg tadalafil per day, 20 mg tadalafil per day, or placebo, for at least 20 days prior to surgery. The drug was discontinued 36 hours before surgery. 35 patients were initially randomized, though 3 withdrew from the study due to back pain or myalgia, likely due to the study drug. 31 patients completed the study. Immunosuppressive cells (monocytic myeloid-derived suppressor cells and Tregs) were evaluated in the blood of the patients before treatment, at the time of surgery, and at least 6 weeks after surgery. The percentage of these immune-suppressive cells in the blood of the tadalafil treated patients was significantly reduced in most cases by the time of surgery. CD8+ T-cells drawn from the blood of the patients at the various time points were then stimulated with tumour lysate-pulsed dendritic cells. Proliferation of CD8+ cytotoxic T-cells significantly increased in most patients treated with taladail, while there was no significant increase in proliferation of CD8+ T-cells from the placebo treated group.

While there was no obvious superiority of either dosing schedule (10 mg versus 20 mg), when the dose response was analyzed in relation to patient body weight, it became apparent that an intermediate dose of tadalafil was more effective than a higher dose, with maximum effect between 0.145 and 0.225 mg of the drug per kilogram body weight. The lessened efficacy of the drug was explained as being due to off-target inhibition of PDE11 that only comes into effect at the higher dose.

Following surgery, paraffin-embedded tumour specimens were evaluated by immunofluorescence. A trend towards fewer immunosuppressive MDSCs in the tadalafil-treated specimens was observed (p=0.09). The activation marker CD69 detected on CD8+ T-cells was significantly increased in patients treated with 10 mg tadalafil, but not in patients treated with 20 mg, again demonstrating superiority of a lower dose of the drug versus a higher dose. When the dose response was again examined relative to patient body weight, patients receiving the lower dose of the drug per kg body weight had significantly fewer MDSCs detected in their tumours. In a retrospective analysis of a different group of patients, the MDSC concentration at the tumour site significantly predicted shorter time to recurrence.

In summary, this study provides clinical validation of previous preclinical studies with mice showing improved survival of tumour-bearing mice after administration of PDE5 inhibitors, an effect linked to the inhibition of myeloid-derived suppressor cells. The present study shows that a lower dose is more effective than a higher dose, likely due to off-target effects of the drug at higher doses. The best results appear to come from a dose of tadalafil in the range of 0.145-0.225 mg per kilogram body weight, equating to 9 – 13.5 mg per day for a 60 kg (132 pound) adult.

Sildenafil – preclinical evidence

Sildenafil has also been shown to stimulate anti-cancer immunity in animal models, primarily through reversing the suppressive capacity of myeloid-derived suppressor cells (MDSCs). A 2006 study [18] demonstrates this impressive and unexpected ability of Viagra in immunocompetent mice bearing subcutaneous tumour implants of colon, mammary, fibrosarcoma, and melanoma cell lines. Sildenafil was given to the mice either in their drinking water, or injected intraperitoneally, at a dose comparable to the upper range of the normal human dose, beginning on the same day as the cancer cell implantation. By day 18-20, tumour size was inhibited by 50-70% in the sildenafil treated mice compared to the untreated controls. This result was also replicated in one model when sildenafil treatment began on day 7 after tumour implant. When immune-knockout mice were given the same treatment, sildenafil had no tumour-suppressing activity, showing that the tumour-suppressing mechanism of sildenafil is related to immune system activity.

Figure 1 Serafini 2006

Figures on the right demonstrate that sildenafil had no effect in mice lacking mature T-cells (Rag-2 knockout mice).

Sildenafil was further found to be synergistic with adoptive vaccine-primed CD8+ T cell immunotherapy. The drug greatly increased CD8+ T cell infiltration into the tumours and also increased their degree of activation.

Following in vitro tests with separate immune cell populations showing predominant drug effects on the myeloid-derived suppressor cell populations, an in vivo test was carried out to confirm the in vitro results. Mice were implanted with mammary cancer cells, half were treated with sildenafil, and were sacrificed 15 days later. Intratumoral MDSCs were obtained, showing reduced IL-4Ralpha (a functional suppressive marker), and downregulated expression and activity of NOS2 and ARG1 enzymes. These enzymes are the primary mediators of MDSC immunosuppressive activity.

Further in vitro studies were carried out showing that the drug could almost completely restore CD4+ and CD8+ T-cell responsiveness when the T-cells were co-cultured with MDSC. These results were replicated in vitro using T cells from multiple myeloma and head & neck cancer patients. Thus, the in vitro studies provide a mechanism for the in vivo observations of increased T-cell infiltration and activity, and smaller tumour size in mice treated with clinically relevant doses of the drug.

This study did not directly address glioma, and it is hoped that these encouraging results will be followed up in glioma studies. As mentioned above, the use of sildenafil as a facilitator of drug transport into brain tumours has been validated in a glioma rat model, and is currently being tested in a clinical trial for recurrent high grade glioma patients.

Vitamin D3 and calcitriol
Preclinical evidence

A paradox sometimes encountered in researching cancer immunotherapy is that the same compound may have 1) immunosuppressive effects in the context of human autoimmune disease or in non tumour-bearing animal studies, and 2) immune stimulating effects in tumour-bearing animals or cancer patients.

A good example of this paradox is vitamin D3, which may increase (immunosuppressive) regulatory T cell function in the context of an autoimmune disease such as multiple sclerosis [10], while increasing effector T cell activity in cancer patients or in vivo models. A series of experiments published in the 1990s showed that vitamin D, injected into mice bearing subcutaneous Lewis lung carcinoma tumours, increases T cell proliferation and activation indirectly, by inhibiting the accumulation of immunosuppressive bone marrow-derived (myeloid) cells.

Lewis lung carcinoma cells (and other types of cancer cells) secrete granulocyte-macrophage colony stimulating factor (GM-CSF), which may increase the presence of immunosuppressive myeloid cells. In one study [11], when mice bearing subcutaneous Lewis lung carcinoma tumours were injected intraperitoneally with vitamin D3 (5 micrograms per kilogram body weight), tumour secretion of GM-CSF was reduced by 75%. This vitamin D3 treatment also reversed the T-cell suppressive activity of bone marrow and spleen cells from tumour-bearing mice. While splenic T-cells from tumour-bearing mice were crippled in their ability to proliferate in response to concanavalin A (a T-cell mitogen), vitamin D3 treatment nearly normalized the T-cells’ ability to proliferate.

Though the tumour growth rate in the mice was only transiently inhibited by the vitamin D treatment, there was a more dramatic effect on metastasis: vitamin D3 treatment reduced the number of metastatic lung nodules by around 75%. In a follow-up experiment, the subcutaneous tumours were surgically removed and vitamin D treatment was started at that time. In the vitamin D treated animals, there was a reversal of immunosuppressive activity in the bone marrow and spleen cells, as seen in the previous experiments. The Lewis lung carcinoma cells used in this study are highly invasive and metastatic. Impressively, after 2 weeks of vitamin D treatment, less than 30% of the mice showed signs of tumour recurrence. In contrast, around 90% of the non-treated mice showed tumour recurrence in the same time period.

Another study [12] by the same group revealed a differential effect of calcitriol (the hormonally active metabolite of vitamin D) on T-cells, depending on whether they were derived from tumour-bearing or non tumour-bearing hosts. The same Lewis lung carcinoma cell line was used in this study. T-cells from normal mice and tumour-bearing mice were exposed to a T-cell receptor (TCR)/CD3 stimulant, plus various concentrations of calcitriol. Normal T-cells’ proliferation was unaffected by lower concentrations of calcitriol and was inhibited by higher concentrations. In contrast, T-cells from tumour-bearing animals were stimulated by a 100 picomolar concentration of calcitriol, which is within the range of calcitriol levels found in the bloodstream. This physiologic dose of calcitriol increased the TCR/CD3 stimulated proliferation of T-cells to the same level as T-cells from non tumour-bearing hosts, essentially reversing their tumour-induced immunosuppressed condition.

Unfortunately, to my knowledge no such studies exist testing the effects of vitamin D3 or calcitriol on the immune status of glioma patients, while clinical trials have been carried out for head and neck cancers.

Clinical evidence

In 2010, results of a small randomized pilot trial involving 32 newly diagnosed head and neck squamous cell carcinoma (HNSCC) patients were published. In this trial [13], patients were randomized, 16 in each group, to receive either oral calcitriol (1,25 dihydroxyvitamin D3) or placebo for three weeks prior to surgical tumour removal. Calcitriol was given at a dose of 4 micrograms per day for three days, followed by a four day rest. After three weeks of calcitriol/placebo treatment, tumours were resected and analyzed for presence of the T-cell markers CD3, CD8 and the activation marker CD69, expressed on lymphocytes and natural killer cells.

The tumours from the calcitriol-treated group showed a 3.1-fold increase in CD4+ T-cells (44 versus 14 per microscopic field) and a 4.4-fold increase in CD8+ cells (75 versus 17 per microscopic field). CD8 is a marker of cytotoxic T lymphocytes, which can be induced to directly destroy cancer cells. Additionally, tumours from the treated group showed a striking increase in cells positive for the immune activation marker CD69 (average 30 per microscopic field), while untreated tumours were either negative or had a few weakly positive cells (average 3 per field).

Patients in both groups were balanced in terms of post-surgery treatment, with 12 in each group undergoing chemotherapy and/or radiotherapy. Mean age was also similar between groups (66 versus 63). All patients were followed up until tumour recurrence. Strikingly, patients in the calcitriol group had a median time to recurrence of 620 days versus 181 days for the placebo group, a 3.4-fold increase. Though this was a small trial, this large difference was statistically significant (p=.048).

A prior clinical study [14] with a very similar design had shown that calcitriol treatment for 3 weeks prior to surgery for head and neck cancer could reduce the tumour population of immunosuppressive CD34+ bone marrow-derived progenitor cells, and increase the numbers of mature dendritic cells. Preclinical studies [15] have also shown a homing of CD34+ hematopoietic progenitor cells to tumours when injected into mice bearing intracranial glioma xenografts. These studies with head and neck cancers may therefore have some relevance for glioma patients.

While supplemental calcitriol is not generally available, this hormone is created in the kidneys from the vitamin D metabolite calcidiol (25, hydroxyvitamin D3). Higher vitamin D levels in the body through exposure to sunshine, or taken in the diet, lead to higher calcidiol levels in the blood, which in turn tends to increase circulating calcitriol. Alfacalcidol (a prescription medication) is directly converted into calcitriol in the liver. Hypercalcemia (an excess of calcium) is the most common side-effect of overly high vitamin D intake.

Selenium (as sodium selenite)

In another study [30] with head and neck cancer patients, 200 micrograms per day of sodium selenite elevated 3 markers of immune functioning after 4 or 8 weeks of supplementation. 30 patients with squamous cell carcinoma of the head and neck were randomized to take either 200 micrograms of sodium selenite per day, or placebo, in the form of oral tablets, beginning on the first day of therapy (surgery or radiation). After 4 weeks of supplementation, lymphocyte proliferation in response to phytohemagglutinin was increased by 121% in the selenite group, versus a decrease of 51% in the placebo group, a statistically significant difference (p=<0.05). After 8 weeks of supplementation, the ability of cytotoxic T lymphocytes to destroy tumour cells was increased by 54.5% compared to baseline in the selenite group, while the same marker was decreased by 23% in the placebo group, also a statistically significant difference. At 8 weeks, in response to alloantigen, lymphocyte proliferation was increased by 2% in the selenite group, versus a decrease of 37% in the placebo group.

In vivo experiments with mice confirmed these findings. Mice were maintained for 8 weeks on a selenium-normal diet, or a selenium-enhanced diet and then injected subcutaneously with syngeneic mouse squamous cell carcinoma cells. In the selenium-normal group, 100% of the mice had detectable tumours by day 8, versus day 17 in the selenium supplemented group, a highly significant difference (p=<0.001). This study refers to previous studies which showed that selenium enhances expression of the various subunits of the interleukin-2 receptor, leading to enhanced proliferation and differentiation of immune precursor cells into cytotoxic effector cells.

Preclinical studies

  • Aspirin
  • Metformin
  • Maitake
  • Ganoderma formosanum (a Taiwanese species of reishi mushroom)
  • Curcumin
  • Withania somnifera (Ashwagandha)
  • Probiotics
  • Caloric Restriction
  • Cimetidine (Tagamet)
  • Low dose Naltrexone and aged garlic extract
  • COX-2 inhibitors
  • Cytomegalovirus and immune suppression

In 2011, Hideho Okada (corresponding author) and coworkers at the University of Pittsburgh published mouse studies [45] showing that low dose aspirin or the COX-2 inhibitor celecoxib could inhibit gliomagenesis and increase mouse survival. Aspirin treatment was effective only when started at day 0, before mice had established tumours. Both aspirin and celecoxib significantly reduced plasma levels of the immunosuppressive prostaglandin PGE2. The effects of aspirin on the immune environment in the mice were then investigated in more deatail. Early aspirin treatment significantly reduced the RNA expression of Ccl2 in the glioma tissue of treated mice. Ccl2 is a chemokine that attracts immunosuppressive myeloid-derived suppressor cells (MDSC). Aspirin-treated mice also had higher RNA levels of Cxcl10 in the tumour environment. Cxcl10 is a chemokine that attracts activated T cells. Aspirin-treated mice also exhibited lower levels of granulocytic myeloid derived suppressor cells in the tumours and bone marrow. Aspirin treatment inhibited the expression of the immunosuppressive molecule Nos2 (inducible nitric oxide synthase) by granulocytic MDSCs in the tumour, spleen and bone marrow, and by monocytic MDSCs within the tumour. Aspirin-treated mice had increased levels of glioma-infiltrating CD8+ T cells with increased effector functions.

The dose of aspirin given to the mice in this study was 10 mg/kg mouse body weight per day. This is likely comparable to low doses of 80 mg aspirin in humans, the dose commonly used for prevention of angina and heart attack in those at risk. Caution: combined use of aspirin and Celebrex increases the risk of gastrointestinal ulceration, due to the simultaneous inhibition of COX-1 and COX-2.


A surprising study [44] appearing in the February 10, 2015 edition of the Proceedings of the National Academy of Sciences showed that various effects on CD8+ T-cell populations may be one of the main anti-tumour mechanisms of the type 2 diabetes drug metformin.

In this study by Japanese scientists, wild-type BALB/C mice were intradermally injected with mouse leukemia cells, followed by administration of metformin in the drinking water on day 7 after tumour inoculation. Most impressively, metformin-treated mice had a gradual elimination of the tumour by approximately day 30, while untreated control mice had progressive tumour growth. After metformin treatment was stopped at day 30, mice who had complete tumour rejection were re-challenged with double the number of mouse leukemia cells. These mice did not develop new tumours upon re-challenge suggesting an immune memory response. When SCID mice lacking T and B cells were injected with leukemia cells and treated with metformin, no tumour inhibiting effect was observed. Depletion of CD8+ T-cells with an anti-CD8 monoclonal antibody eliminated the anti-tumour effect of metformin in treated mice, while treatment with an anti-CD4 antibody had no such effect (see figure 1B below). This experiment demonstrated the specific importance of CD8+ T-cells in the metformin-induced tumour shrinkage.

Figure 1 Eikawa immune effects of metformin

Figure A, treatment of mice with metformin in the drinking water led to complete tumour rejection in mice. Metformin treatment of SCID mice lacking T-cells had little to no effect on tumour growth. Figure C-E, metformin treated mice had increased number of tumour-infiltrating lymphocytes at day 10 and 13. Figure F, metformin treatment reduced the apoptosis (cell death) of CD8+ lymphocytes.

Different doses of metformin in the drinking water (5 mg/mL down to 0.2 mg/mL) were tested in the same mouse model. Metformin dose-dependently inhibited tumour growth even at doses of 0.2 mg/mL in the drinking water, which leads to drug levels in mouse plasma that is certainly clinically achievable in humans with standard drug dosing.

Metformin treatment was then tested in four additional syngeneic mouse tumour xenografts: renal cell
carcinoma, 3LL non small cell lung carcinoma, Colon 26 intestinal carcinoma, and 4T1 breast cancer. As with the leukemia xenografts, metformin also induced complete tumour rejection in the renal cell carcinoma model. Significant, but incomplete tumour inhibition was observed in the remaining 3 models.

Further studies showed that metformin treatment reduced apoptosis (cell death) of CD8+ tumour-infiltrating lymphocytes in treated mice, and increased the ratio of T-effector memory to T-central memory cells (TEM/TCM ratio). The investigators concluded that T-effector memory cells are more involved in tumour rejection than T-central memory cells. In the discussion the authors state that “Our used model systems comprised highly immunogenic tumors, and it is unclear whether metformin would have the same effect on less immunogenic tumors.”

This study is significant in that tumour inhibition was observed even at the lowest dose of metformin given to mice in the drinking water. No tumour inhibition was observed in SCID mice lacking T cells. This has profound implications in that it suggests the anti-cancer effects of metformin, at least for more immunogenic tumours, could be more due to this immune mechanism than due to direct effects on tumour cells or changes in blood glucose/insulin. We hope that this study will lead to many further studies of the immunological effects of metformin, especially in human cancer patients.

Maitake Mushroom (Grifola frondosa)

The immune stimulating properties of many mushroom species are well known, though the power of these mushrooms to fight cancer has been insufficiently tested in controlled clinical trials. In the 1990s, Hiroaki Nanba, a Japanese scientist, conducted informal trials of maitake in patients with various types of cancer. He documents that over 35% of brain tumours in his study responded to maitake alone with at least symptomatic improvements or direct tumour-suppressing effects [1].

The most active cancer-fighting component of Maitake is called “D-fraction”. One of the best recent studies of the interaction of maitake D-fraction with the immune system was published by Yuki Masuda, along with Hiroaki Nanba and others in 2012. In this study [2], mice were implanted in the flanks with mammary carcinoma or colon adenocarcinoma cells and given oral maitake D-fraction every day for 16-20 days. Maitake D-fraction treatment dose-dependently inhibited tumour growth, leading to significantly smaller tumour volumes throughout the study period, compared with the untreated control animals.

Figure 1 Masuda 2013

Next, the precise mechanisms for this anti-cancer effect were investigated. The colon adenocarcinoma model was used, and a dose of 20 mg/kg body weight maitake D-fraction was given orally to mice for 20 days. It was found that maitake D-fraction (MD-Fraction) was taken up by macrophages and dendritic cells in the gut-associated lymphoid tissue (GALT), and from there was transported to the spleen by day 3.

Effects of D-fraction on Cytotoxic T cells and cytokine balance

On day 20 after inoculation with colon cancer cells, MD-Fraction treated mice had significantly increased numbers of CD8+ cytotoxic T cells, B cells, and natural killer cells in their spleen, while there was a dramatic decrease in numbers of myeloid cells. MD-fraction treatment increased interleukin-12 and interferon-gamma mRNA levels 3.9- and 2.5-fold. MD-Fraction treated mice had more interferon-gamma expressing CD8+ cells in spleens and tumours. The chemokines CXCL9 and CXCL10, which attract cytotoxic T cells and promote their infiltration into tumours, were increased 4.7- and 3.2-fold in tumours of MD-Fraction treated mice in comparison with untreated control mice. Further, there was a 2.6- and 2.9-fold increase in tumour infiltration of CD4+ and CD8+ T-cells in the treated mice. The tumour inhibiting effect of MD-Fraction is likely mediated by the increased T-cell infiltration, as nude mice lacking T-cells gained no benefit from maitake in terms of tumour inhibition.

Activation of dendritic cells

In vitro exposure of dendritic cells to MD-Fraction led to an increased expression of the maturation markers CD80, CD86 and MHC II, required for effective antigen presentation. MD-Fraction also increased the expression of these molecules in macrophages.

Regulatory T-cell inhibition

When tumours from MD-Fraction treated animals were removed and analyzed after 20 days, there was found to be a significant decrease in the percentage of immunosuppressive Tregs (0.5% versus 0.9% in the untreated tumours).

Myeloid-derived suppressor cell inhibition

Myeloid-derived suppressor cells (MDSCs) are immune-suppressive myeloid cells which repress T-cell and natural killer cell functions by secreting substances such as arginase 1. In MD-Fraction treated mice, the percentage of MDSCs in tumours was 5.8% after 20 days, compared with 14.4% in the untreated mice. There was also a significant reduction in arginase 1 and other immunosuppressive cytokines such as interleukin-10 and transforming growth factor-beta in the treated tumours.

In short, this study proves that orally administered maitake D-fraction is a broad spectrum immune stimulant which promotes the cytotoxic, anti-cancer arm of the immune system in multiple ways, while simultaneously inhibiting the immunosuppressive, tumour-promoting arm of the immune system. This ability translated into significantly slowed tumour growth in immunocompetent mice implanted with mammary and colon cancer cells. A dosage of 100 mg of maitake D-fraction daily has been used previously in maitake studies in human cancer patients in Japan.

Ganoderma formosanum mushroom extract

Ganoderma formosanum is a species of reishi (also called lingzhi) mushroom, native to Taiwan. Reishi species are very commony used medicinally in east Asia. A study [43] with tumour-bearing mice demonstrated the anti-cancer immunostimulatory powers of a polysaccharide extract from Ganoderma formosanum, called PS-F2. Of note, the monosaccharide profile of this extract differs from extracts of other Ganoderma (reishi) species.

Intraperitoneal injections of the PS-F2 extract significantly inhibited tumour growth in mice bearing subcutaneous colon carcinoma and melanoma tumours. When SCID mice lacking functional T-cells and B-cells were treated with PS-F2 extract, there was no effect on tumour growth. When the PS-F2 was given orally to mice bearing sarcoma tumours (S180), beginning on the day before tumour implantation, tumour regression was seen after day 15. Further experiments demonstrated that the anti-tumour effect of the mushroom extract was likely due to activation of CD4+ and CD8+ T-cells, as well as the production of anti-tumour antibodies in the serum.


In addition to any direct anti-cancer effects, curcumin has been shown to be a suppressor of myeloid-derived suppressor cells (MDSCs). Glioblastoma patients, and likely other glioma patients, have elevated levels of circulating MDSCs compared with healthy age-matched controls [3], and this contributes to the tumour-induced immunosuppression found in these patients.

Curcumin effects on myeloid-derived suppressor cells

In one experiement [4], immunocompetent Balb/c mice were implanted in the flank with syngeneic CT26 colon cancer cells. After tumours reached 100 cubic millimetres, animals were treated with either oral curcumin as 2% of their diet (this is far more curcumin than a human is likely to ingest), or intraperitoneal injections of curcumin at a dose of 50 mg per kilogram mouse body weight. In both groups of treated mice, tumour volumes and weights were significantly less than the tumours of the untreated control mice by day 22 or 28.

Figure 1 Tu 2012 Curcumin (large)

The percentage of MDSCs in both the spleens and tumours of curcumin treated mice were lower than in the untreated control mice. There was also an increase in apoptosis (cell death) of MDSCs in the spleens of the treated mice. Both the direct anti-cancer effects and the immunological effects of curcumin may be due to its ability to inhibit pro-carcinogenic and immunosuppressive transcription factors such as STAT3 and NF-kB. In this experiment, in vitro concentrations of 12.5 micromolar (likely achievable in vivo) inhibited expression of both STAT3 and NF-kB.

Curcumin Effects on T-helper cells, cytotoxic T cells, cytokine balance, and Tregs in tumour-bearing mice

Even more compelling evidence for the immune-stimulating effect of curcumin in tumour-bearing mice comes from a study by the Division of Molecular Medicine, Bose Institute, Kolkate India, and published in Cellular & Molecular Immunology in 2010 [5]. In this experiment, immunocompetent Swiss albino mice were implanted intraperitoneally with Ehrlich’s ascites (mammary) carcinoma cells. Seven days post-implantation, 10 mice in the experimental arm were fed oral curcumin at the moderate dose of 50 mg per kilogram body weight. After 21 days, curcumin fed-mice were compared with the untreated control mice.

Untreated tumour-bearing mice had diminished populations of CD4+ T-helper cells and CD8+ cytotoxic T lymphocytes (CTLs) in circulation and lymph nodes compared to non tumour-bearing mice. Curcumin-treated mice had normalized CD4+ and CD8+ populations, comparable to the non tumour-bearing mice. Curcumin-treated mice had significantly elevated levels of CD4+ and CD8+ cells in tumours compared with the untreated tumour-bearing mice.

Memory T cells are antigen-experienced T cells which accumulate over an individual’s lifetime, and consist of central memory and effector memory T cells. In this experiment, untreated tumour-bearing mice had diminished populations of both types of memory T cells in circulation and lymph nodes compared with non tumour-bearing mice. Curcumin-treated tumour-bearing mice had normalized populations of memory T cells, and significantly elevated levels of memory T cells in tumour tissue compared with untreated tumour-bearing mice.

Interferon-gamma (IFN-gamma) is one of the major Th1 type cytokines promoting cellular-mediated immunity, while interleukin-4 (IL-4) is a characteristic Th2 type cytokine. Th1 polarization is necessary for effective anti-cancer immune responses. In this experiment, untreated tumour-bearing mice had diminished interferon-gamma expressing CD4+ and CD8+ T cells in circulation and lymph nodes compared with non tumour-bearing mice. Curcumin-treated tumour-bearing mice had normalized levels of interferon-gamma expressing T cells and significantly increased interferon-gamma expressing T cells in the tumour tissue compared with untreated tumour-bearing mice. The reverse pattern was seen for interleukin-4. In response to a T-cell antigen receptor stimulus, T cells from tumour-bearing mice failed to proliferate, while T cells from curcumin-treated tumour-bearing mice had a normal proliferative ability.

Regulatory T cells (Tregs) are immunosuppressive cells which express cytokines such as interleukin-10 and transforming growth factor-beta (TGF-beta), potent immunosuppressive molecules. Treg populations were elevated in the circulation and lymph nodes of untreated tumour-bearing mice. Curcumin normalized the percentage of Tregs in the treated tumour-bearing mice, and significantly lowered the percentage of Tregs at the tumour site compared with untreated tumour-bearing mice. Curcumin treatment also normalized the expression of TGF-beta and IL-10 by Tregs in the circulation, lymph nodes, and tumour.

The immune-stimulating effect of curcumin was possibly at least partially due to direct interaction with tumour cells: in vitro, T cells cultured with supernatant (liquid) from tumour cells pre-exposed to curcumin were more effective at tumour cell killing versus T cells cultured with supernatant from tumour cells unexposed to curcumin. Other studies have shown that curcumin is an inhibitor of STAT3, an immune-suppressive transcription factor often upregulated in cancer cells [6].

Most importantly, at 21 days post-implantation, untreated mice hosted an average of 390 million tumour cells, while curcumin-treated mice hosted an average of 20 million, a 20-fold reduction. At 21 days, all curcumin treated mice were still alive, while half of the untreated mice had died. Like Maitake D-fraction, curcumin has multiple activating effects on the anti-cancer immune system in vivo in tumour-bearing animals. It is likely that this effect is context-dependent, as curcumin may have immunosuppressive effects in non-tumour bearing animals, and is also useful in the context of auto-immune diseases, where it dampens down pathological immune activation.

Withania somnifera (ashwagandha)

Withania somnifera is a plant belonging to the Solanaceae (nightshade) family, also known as ashwagandha in the Ayurvedic tradition, and as Indian ginseng and winter cherry. It is cultivated in drier regions of India and Nepal. Withanolides derived from root and leaf extracts have adaptogenic qualities and may be used for stress relief, sleep improvement, helping with fatigue, and stimulating immune responses.

In the lab, Withania also slows growth of tumours in experimental animals. One study [24] from the Indian Institute of Integrative Medicine showed that a Withania formulation containing root and leaf extracts in a 1:1 ratio dose-dependently inhibited Ehrlich ascites tumours in Swiss albino mice. The formulation contained about 4% withanolides/glycowithanolides on a dry weight basis, including 0.8% withanone, 0.7% withanolide A and 0.6% withaferin A.

After intramuscular injection of the Ehrlich ascites cells, the mice were given oral Withania formulation at doses of 100, 200, or 400 mg per kilogram mouse body weight per day, for either 9 or 21 days. Tumour weight was dose-dependently reduced by Withania formulation, with the 400 mg/kg dose leading to a 51% growth inhibition after 21 days. In comparison, etoposide chemotherapy was only slightly more effective, with a 59% growth inhibition after 21 days.

Withania treatment also improved immune responsiveness compared to untreated controls, leading to increased blood levels of interferon-gamma (maximum in the 200 mg/kg group) and interleukin 2 (maximum in the 100 mg/kg group). Percentages of CD4+ and CD8+ T-cells were highest in the 100 mg/kg dosed group, and immune co-stimulatory molecules were also highest in the 100 and 200 mg/kg dosed groups. Phosphorylated STAT3 (which has immunosuppressive effects) was lowest in the tumour tissue of the 400 mg/kg dosed group.

In chronic toxicity studies, oral doses of 500, 1000 and 1500 mg/kg body weight were given to Wistar rats for 6 months: no toxicity or other abnormalities were found.

In this study, maximum tumour inhibition was achieved at the highest dose of 400 mg/kg mouse body weight. The particular formula used in this study used root and leaf extract in a 1:1 ratio, with a total withanolide content of about 4%. A commercial root and leaf preparation called Sensoril, available in many different brands of ashwagandha, claims a minimum glycowithanolide content of 10%, the highest bioactive content on the market. Glycowithanolides are said to be more bioactive and adaptogenic compared to free withanolides in Withania extracts. It is not yet clear which fraction is the most potent in terms of cancer therapy. A clinical human trial of Sensoril gave doses of 250 mg twice per day, 125 mg twice per day, or 125 mg once per day. Clinical benefits were about equal with all 3 dosing regimens. As seen in the preclinical cancer study above, the anti-cancer effects of Withania extract may be more dose-dependent than the improvement of stress and fatigue seen in the human clinical trial at all dose levels.

It must be kept in mind that many ashwagandha products on the market, such as Sensoril, have not been designed as anti-cancer agents, and the optimal blend of withanolides/glycowithanolides to fight cancer is not yet established. As an example, Sensoril claims to minimize the content of withaferin A, which has shown potency in preclinical cancer studies, including glioma studies. Nevertheless, patients might benefit from the stress and fatigue reducing, adaptogenic qualities of a supplement such as Sensoril.

Different withanolides predominate in root versus leaf extracts

Two studies by the same group analyzed the withanolide content of pure root versus pure leaf extracts of Withania, and the Th1 immune-stimulating properties of each. In the first study [25], 8 withanolides and 3 glycowithanolides were quantified in standardized Withania root extract. Total withanolide/glycowithanolide content was about 2% and Withanolide-A was found to be the predominant compound, making up 1.34% of the extract. Withaferin-A composed only 0.017% of the extract. Though not a cancer study, Withania root extract proved to have a Th1 immune-stimulating effect when administered orally to mice for 15 days, with maximum stimulation of Th1 cytokines interferon-gamma and interleukin-2 at the dose of 30 mg/kg mouse body weight. The dominant withanolide in the root extract, withanolide-A, was also shown to have the same Th1 skewing effect as a pure compound.

In the second study [26], 11 withanolides/glycowithanolides were analyzed in a standardized leaf extract. Total withanolide/glycowithanolide content in the leaf extract was around 8% (much higher than the 2% in the root extract). The specific withanolide composition was quite different in the leaf extract, with three being predominant: 2,3 dihydro-3-sulphonile withanone (WSL-2) at 3.25%, Withaferin-A at 1.9%, and Withanone at 1.3%.

In vitro, Withania leaf extract stimulated the secretion of interferon-gamma (a Th1 cytokine) by mouse splenocytes dose-dependently. In vivo, small doses (25 mg/kg) of oral leaf extract doubled the population of CD3+, CD4+ and CD8+ T-cells, and also increased the CD19+ B-cells population. Similarly, 25 mg/kg leaf extract also doubled the expression of immune co-stimulatory molecules CD80 and CD86 on antigen presenting cells.

When individual withanolides from the leaf extract were tested in vitro, only WSL-2 (2,3 dihydro-3-sulphonile withanone) was effective in dose-dependently enhancing interferon-gamma expression. This compound also happens to be the predominant withanolide in the standardized leaf extract. This withanolide also stimulated IL-12 production by mouse peritoneal macrophages in vitro, skewing them to a type-1 phenotype. In cancer, macrophages are typically skewed to an immunosuppressed type-2 phenotype.

In summary, both root and leaf extracts of Withania somnifera have potent Th1 immunostimulatory activity in mice, attributable to withanolide-A in the root extract, and to WSL-2 in the leaf extract. However, as clearly demonstrated in reference 24 (described above), Withania has maximum tumour-inhibiting effect at high doses, beyond those which lead to maximum immune-stimulation. This implies that Withania also has direct anti-tumour activity in addition to activation of the immune system. Several in vitro studies [27, 28] have revealed that Withaferin-A is by far the most potent withanolide against glioma cell lines, however its ability to penetrate the brain in sufficient quantities appears limited, as treatment of mice with experimental brain tumours required relatively high doses to achieve therapeutic effect [28] or combination with an experimental STAT3 inhibitor [29].

Probiotics: Lactobacillus acidophilus and L. casei

Two studies by Iranian scientists demonstrated the immune stimulating effects of Lactobacteria strains and its therapeutic benefit in immunocompetent mouse models of breast cancer.

In the first study [22], spontaneous mouse breast tumours were transplanted into the flanks of the BALB/c test mice following a 14-day pre-treatment with 0.5 mL/day oral L. acidophilus suspension. Following the tumour implantation, L. acidophilus treatment was continued until day 30. Compared with the untreated mice, mice receiving L. acidophilus bore tumours which were 34% smaller in volume. Spleen cells from the treated mice were more proliferative in response to tumour antigen compared to spleen cells from untreated mice. Spleen cells from treated mice also secreted higher levels of IL-12 (a type 1 immune cytokine) and lower levels of transforming growth factor beta (TGF-beta, an immunosuppressive cytokine) in response to tumour antigen.

In the second study [23], mice were treated as in the first study except that Lactobacillus casei spp. casei was used in place of acidophilus. Tumour volume was reduced by 46% compared to tumours in the untreated control mice. Spleen cells from treated mice produced significantly higher levels of the Th1 cytokines IL-12 and interferon gamma (IFN-g). Natural killer cells from treated mice demonstrated increased cytotoxic abilities. Most importantly, survival was prolonged in the treated mice versus the untreated control mice: median survival in the control group was around 35 days, while median survival was over 65 days in the treatment group (according to figure 3).

L. acidophilus is a popular probiotic and may be purchased as a supplement, and is also found in probiotic dairy products such as yoghurt. L. casei is found in ripening cheddar cheese, and probiotic beverages such as Yakult (commercial brand, made in Japan) and Actimel yoghurt (known as DanActive in North America).

Caloric restriction and anti-cancer immunity in mice

It has long been observed in animal experiments that calorie-restricted diets can lead to increased longevity, and one of the possible mechanisms is a tonic effect on the immune system. A mouse experiment published in 1991 showed that caloric restriction can suppress carcinogen-induced tumour incidence and growth rate by stimulating anti-cancer T-cell responses [16]. In this experiment, mice were divided into an unrestricted diet group (fed 5 grams of food per day) and a restricted diet group (fed 3 grams per day). After 8 weeks of the separate diets, the mice were injected with the potent carcinogen 3-Methylcholanthrene (MC). At day 114 after MC injection, all mice were sacrificed and examined. All 22 mice in the unrestricted-diet group had confirmed neoplastic (cancerous) tumours. While the majority of diet-restricted mice also had putative tumours at this time, half of these tumours proved to be simply cases of lipid necrosis, perhaps due to the corn oil used as vehicle for the MC injections. Neoplastic tumour incidence after 114 days was therefore 100% in the diet-unrestricted group but only 50% in the diet-restricted group. The tumours in the restricted group also took longer to form and were slower growing, not changing significantly in diameter between day 94 and day 114, while tumours in the unrestricted diet group progressively increased in size.

Next, the immune cell composition was compared between the two groups. Diet-restricted mice had a dramatically increased percentage of Thy1+ L3T4+ T-helper cells as a proportion of splenic T cells. Spleen cells from diet-restricted mice showed the same degree of response when stimulated with concanavalin A (a T-cell mitogen), and the same or higher response to interleukin-2 (IL-2), when compared with normal mouse spleen cells. In contrast, spleen cells from diet-unrestricted tumour-bearing mice were suppressed, with markedly decreased response to IL-2.

A second experiment was then conducted to determine the timing of immune system effects in response to diet. Again mice were divided into diet-restricted and unrestricted groups and later injected with the carcinogen MC. At five weeks after MC, before any mice had developed tumours, there was already a significant difference in immune response between the two groups. T-cells from diet-restricted mice had markedly increased response to stimulation with Con A, IL-2 or alloantigens, compared to T-cells from diet-unrestricted mice. While dietary restriction suppressed natural killer cell activity, CD8+ cytotoxic T lymphocyte (CTL) activity in response to alloantigens was strongly increased in the diet-restricted group. This difference was observed at all three time points, including the early time point before tumours were apparent, and is therefore related directly to the dietary difference rather than the increased tumour burden in the diet-unrestricted group. The increased proportion of T-helper cells, and increased cytotoxic T-cell responsiveness in the diet-restricted group is likely one of the main mechanisms of reduced carcinogenesis in these mice.

In the second experiment, when mice were sacrificed at 15 weeks post MC injection, all of the diet-unrestricted mice had tumours, with an average diameter of 9.5 mm. In contrast, only 25% of the diet-restricted mice had formed tumours, and in those that did, the average tumour diameter was only 1.8 mm, an 80% reduction compared with the unrestricted group.

What does this mean for humans? Mouse metabolism is quicker than human metabolism, and a 40% caloric restriction in mice does not translate to a 40% caloric restriction in humans. The actual degree of weight loss is likely more readily translatable to the human scenario. The mice in the diet-restricted group in this experiment started off at around 22 grams, and gradually lost weight over the next 150 days (about 5 months), at which time their average weight was about 16 grams (these estimates are extracted from a graph shown in the study). In other words, these mice lost about 25% of their body weight over this time. Of course, humans vary widely in how much weight they can afford to lose safely. A gradual loss of weight stabilizing in the low-normal range is probably a safe approach that could have significant benefits for anti-cancer immunity.

Cimetidine (Tagamet)

Cimetidine (trade name Tagamet) was introduced in the late 1970s and was a very popular drug in treating heartburn and peptic ulcers. Its mechanism of action is inhibition of histamine H2 receptors, which are linked to the production of gastric acid by parietal cells of the stomach. Though largely superseded by newer proton pump inhibitors for the treatment of gastric disorders, cimetidine is still available by prescription and over-the-counter.

The clinical anti-cancer effects of cimetidine were noted as far back as 1988, when gastric cancer patients taking the drug were noted to have improved survival [31]. Anti-cancer activity of cimetidine in preclinical studies was observed as far back as 1981 [32, 33], when growth of tumour grafts (Lewis lung carcinoma and lymphoma) in mice was inhibited by oral administration of the drug. In these mouse studies, the anti-cancer effect of cimetidine was linked to effects on the immune system, specifically the inhibition of immune suppressor cells. Since these early studies, there have been numerous preclinical and clinical reports of the immune stimulating effects of cimetidine including increased interleukin-12 production [34], increased antigen-presenting capacity of dendritic cells in colorectal cancer patients [35], and increased tumoral infiltration of lymphocytes in gastrointestinal and colorectal cancer patients [36, 37].

A study [38] published late in 2012 by Chinese investigators demonstrated that perhaps the primary immune system effect of cimetidine in tumor-bearing animals is the inhibition and apoptosis of myeloid-derived suppressor cells (MDSC), which play a large role in cancer-associated immunosuppression. In this study, immunocompetent mice were implanted with syngeneic Lewis lung carcinoma cells. On the same day of tumour implantation and every other day until day 14, mice were treated intraperitoneally with a lower or higher dose of cimetidine. By day 32, the tumour size was dose-dependently inhibited in the two groups treated with cimetidine relative to untreated controls. Similarly, survival time was dose-dependently increased in the cimetidine treated groups. The higher dose group of mice had a median survival time of just under 60 days, while the untreated control mice had a median survival of just over 30 days.

Figure 1 Zheng 2013 Cimetidine

In vitro, cimetidine had no influence on the proliferation, survival, migration or invasion of the Lewis lung carcinoma cells, suggesting that the effect of cimetidine in the mice was due to indirect rather than direct effects on the tumour.

The influence of cimetidine on the immune activity of the mice was then studied. The proportion of immunosuppressive MDSCs in spleens, and frequency of MDSCs in blood and tumours of the mice were significantly reduced in the cimetidine-treated mice. In vitro, MDSCs strongly suppressed T-cell proliferation and production of interferon-gamma, while the addition of cimetidine reversed these suppressive effects. Further studies in vitro showed that cimetidine induced apoptosis (death) of MDSCs selectively, with no effect on T-cell apoptosis. Though the histamine H2 receptor was expressed on the surface of MDSCs, a different H2 receptor antagonist, famotidine, did not have the same apoptotic effect on MDSCs as cimetidine.

In light of these results, and the results of other studies going as far back as 1981 showing cimetidine’s strong immunomodulation, the use of athymic mice in xenograft models does not demonstrate the full potential of cimetidine as an anti-cancer agent. Indeed, a 2005 study [39] showed that cimetidine as a single agent was entirely lacking activity in nude (athymic) mice bearing U373 glioblastoma xenografts. In combination with temozolomide, cimetidine had a modest chemosensitizing effect, though with no improvement of median survival time over temozolomide alone. A syngeneic, immunocompetent model would have likely shown a more significant anti-tumour effect of cimetidine, possibly as a single agent, as seen in the previously described study involving Lewis lung carcinoma [38].

Combination therapies involving cimetidine added to other drugs must take into account the fact that cimetidine is a general inhibitor of many cytochrome P450 (CYP) enzymes which are fundamental to drug metabolism in the liver. Consequently, plasma levels of other drugs may be increased when taken together with cimetidine, and dosing should be adjusted accordingly. The standard clinical dose of cimetidine for duodenal and gastric ulcers and for gastro-oesophageal reflux disease is 800 mg once daily, or 400 mg twice daily. This dose has also been used by glioblastoma patients using cimetidine as a repurposed drug as part of their tumour therapy.

Low-dose Naltrexone plus garlic effective in immunocompetent tumour-bearing mice

Naltrexone is an opioid receptor blocker, which when taken in a single very low daily dose, causes a temporary blockade of opioid receptors lasting several hours. To compensate for this blockade, cells in the body increase expression of opioid receptors and production of the body’s endogenous opioids, primarily opioid growth factor (also known as met-enkephalin). Despite its “growth factor” designation, the increased activity of opioid growth factor following low dose naltrexone intake has been shown to have an inhibitory effect on cancer growth. However, many of the preclinical studies of low dose naltrexone in tumour-bearing animals have been performed using immunodeficient xenograft models [19], and the potentially complex effects of endogenous opioids on the immune system was therefore not modeled in these studies.

In 2013, a group of Iranian investigators published a study [20] of low dose naltrexone (LDN) and aged garlic extract (AGE), either alone or in combination, using an immunocompetent syngeneic murine model of fibrosarcoma. Low dose naltrexone and aged garlic extract were both administered intraperitoneally once every three days.

Median survival times were calculated for groups of tumour-bearing mice receiving garlic extract, low dose naltrexone, or both combined, and compared with untreated mice. Garlic extract increased median survival by 61%, LDN increased median survival by 55%, and the combination increased median survival by 155% (in other words a 2.55-fold increase, more than doubling mean survival time). Both AGE and LDN delayed the time to tumour appearance, with the combination treatment delaying tumour appearance most significantly (all mice in the untreated group had tumours by day 14 post-implantation, while none in the combination treatment group showed tumour presence at this time). Similarly, AGE and LDN had approximately equal effects on reducing tumour volumes at any given time, while the combination treatment showed the most dramatic effect on slowing tumour growth.

Figure 1 Naltrexone plus aged garlic

Next, the effect of these treatments on immune cells was investigated. Splenocytes from the treated mice were tested for cytotoxicity against the fibrosarcoma cells in vitro. While both AGE and LDN (in that order) increased the cytotoxicity of splenocytes compared to splenocytes from untreated control mice, only the combination treatment effects reached statistical significance. When splenocytes from the mice were exposed to fibrosarcoma cell lysate, cells from LDN treated mice had slightly higher interferon-gamma production versus controls, though the increase was statistically insignificant. Splenocytes from AGE treated mice showed a statistically significant increase in interferon-gamma production. Interferon-gamma production by splenocytes from combination treated mice was dramatically and synergistically increased.

In summary, this study provides preclinical proof that low dose naltrexone indeed stimulates anti-cancer immune responses in an immunocompetent fibrosarcoma model. While aged garlic extract seemed to have a more profound effect on immune cell cytotoxic potential, individual treatment with either garlic extract or low dose naltrexone had roughly equal effects on survival prolongation and tumour growth inhibition. Most importantly, the combination of aged garlic extract with low dose naltrexone appeared to have a synergistic beneficial effect. The administration of these two agents in this study was by injection, rather than by mouth. Therefore, the oral doses required to achieve comparable results in humans cannot be determined from this study. Low dose naltrexone at 3-10 mg per day in adult humans is the recommended oral dose, with 4.5 mg once per day perhaps the most common dosing. Opioid growth factor (met-enkephalin), which is stimulated by daily low dose naltrexone, appeared to have efficacy in a phase II trial for advanced pancreatic cancer [21].

Aged garlic in the mouse study was prepared by mincing garlic bulbs and storing in anaerobic conditions for 8 months. The aged garlic was then crushed and homogenized in distilled water. The homegenate was then filtered, centrifuged, and further diluted in distilled water. The final preparation was administered to the mice by injection. In other words, the preparation of garlic in this study was fairly complex, and not comparable to oral consumption of whole garlic.

As mentioned above, one of the main endogenous opioids with increased production following low dose naltrexone is met-enkephalin (also known as methionine enkephalin, and as opioid growth factor). A Chinese study published online in August 2016 examined the effects of met-enkephalin on microglial cells in culture [46]. As explained in this study and elsewhere, microglia (the resident macrophages of the nervous system) and macrophages infiltrating from the systemic circulation are actively recruited into tumors, polarized to a tumor-promoting M2 phenotype and away from a tumor-fighting M1 phenotype, and can make up as much as 30% of a GBM tumor. The investigators found that at the optimal concentration of met-enkephalin (1 picomolar), M1-type cytokine production and surface proteins were increased in the microglia, including interleukin-12, TNF-alpha, CD86, CD40, and iNOS. In contrast, M2 cytokines and markers were not affected, including interleukin-10, TGF-beta, CD163, and arginase. Phagocytosis (a main function of M1 macrophages) and cytotoxicity towards U87 glioblastoma cells was increased by met-enkephalin treatment. Thus, met-enkephalin (opioid growth factor), an endogenous opioid whose production is increased in humans following transient opioid receptor blockade by low dose naltrexone, may aid glioma patients by reverting tumor-associated microglia to an M1 anti-tumor phenotype.

View common questions and answers about the use of low dose naltrexone at LDN Science. Especially interesting are interviews with Dr. Ian Zagon, who discovered the clinical benefits of naltrexone in very low doses.

The effective dose of low dose naltrexone ranges from 2.5 – 10 mg, with the most common dose being 4.5 mg daily. LDN may be taken in the morning or evening. Some individuals may experience sleep disturbances (such as nightmares) caused by LDN and these people may choose to take their daily dose in the morning.

COX-2 inhibitors

COX-2 is an enzyme that helps convert arachidonic acid (a 20 carbon omega-6 fatty acid) into products such as prostaglandin E2 (PGE2), which is essential in the process of cancer-induced immunosuppression. Please see the evidence in favor of COX-2 inhibitors such as Celebrex as an addition to immunotherapy on the Immunotherapy page.

Human cytomegalovirus and immune suppression

The role of the common herpesvirus cytomegalovirus (CMV) in glioma genesis and/or progression is still controversial, as is evidence for the presence of cytomegalovirus proteins and nucleic acids in a large proportion of human glioblastoma samples. One study [7] found the CMV protein IE1-72 in 21 of 21 glioblastoma samples, 9 of 12 (75%) anaplastic glioma samples, and 14 of 17 (82%) lower grade glioma samples. Since the CMV-glioma connection was first made by Charles Cobbs (currently of the Ivy Center / Swedish Neuroscience Institute in Seattle) and coworkers in 2002, futher work has confirmed a role for CMV in promoting tumour invasion, angiogenesis, activation of pro-tumour receptor tyrosine kinases (such as platelet-derived growth factor receptor – PDGFR), and cell cycle progression.

Furthermore, a 2011 study [8] demonstrated a role for CMV in converting monocytes to a tumour-promoting M2 phenotype. First, 5 human glioblastoma samples were tested for the CMV protein pp65. The samples tested positive for pp65, which was also found in the tumour-associated macrophages and microglia, but not in T cells or B cells. Next, a panel of four glioma stem-cell lines was tested for CMV activity, and all were found positive for CMV proteins pp65, IE1, and US28 as well as CMV interleukin-10 (CMV IL-10), but not human IL-10.

When peripheral blood monocytes from GBM patients or healthy donors were exposed to CMV IL-10, CMV IE1 expression in the monocytes was induced, suggestive of an an increase in CMV activity in the monocytes. When blood monocytes from healthy donors were cultured with CMV IL-10, the monocyte expression of MHC II and CD86 (both required from antigen presentation and T cell activation) was decreased, while the T cell inhibitory molecule B7H1 (also known as PD-L1) was increased, along with the immunosuppressive and pro-invasive molecules pSTAT3, TGF-beta1, and VEGF.

Next, glioma stem cells were exposed to supernatant liquid from the CMV IL-10 treated monocytes, causing a 40-fold increase in migratory ability compared with glioma stem cells treated with supernatant from untreated monocytes. This was perhaps due to the production of MIP-2 (which induces mobilization of stem cells) by the CMV IL-10 treated monocytes, as well as the production of TGFbeta1 (which also induces glioma motility) by the monocytes.

In summary, this study showed that CMV-infected glioma stem cells secrete CMV IL-10, which both activates CMV in monocytes and converts the monocytes from an M1 phenotype, capable of antigen presentation and T cell activation, to an immunosuppressive, pro-invasive M2 phenotype, producing tumour-promoting pSTAT3, TGFbeta1 and the angiogenic growth factor VEGF.

Anti-cytomegaloviral drugs such as Valcyte (valganciclovir) and Vistide (cidofovir) [9] are under investigation as potential anti-glioma therapies, with many patients supplementing with Valcyte despite the lack of definitive prospective clinical trials, which will hopefully be forthcoming.

  1. Maitake D fraction: healing and preventive potential for cancer. Nanba. 1997.
  2. Oral administration of soluble beta-glucans extracted from Grifola frondosa induces systemic antitumor immune response and decreases immunosuppression in tumor-bearing mice. Masuda et al. 2013.
    READ ABSTRACT (email me for PDF copy)
  3. Myeloid-derived suppressor cell accumulation and function in patients with newly diagnosed glioblastoma. Raychaudhuri et al. 2011.
  4. Curcumin induces the differentiation of myeloid-derived suppressor cells and inhibits their interaction with cancer cells and related tumor growth. Tu et al. 2012.
  5. Curcumin reverses T cell-mediated adaptive immune dysfunctions in tumor-bearing hosts. Bhattacharyya et al. 2010.
  6. Dietary curcumin attenuates glioma growth in a syngeneic mouse model by inhibition of the JAK1,2/STAT3 signaling pathway. Weissenberger et al. 2010.
  7. Detection Of Human Cytomegalovirus In Different Histological Types Of Gliomas. Scheurer et al. 2008.
  8. Glioma-associated cytomegalovirus mediates subversion of the monocyte lineage to a tumor propagating phenotype. Dziurzynski et al. 2011.
  9. Cidofovir: a novel antitumor agent for glioblastoma. Hadaczek et al. 2013.
  10. Vitamin D Status Is Positively Correlated with Regulatory T Cell Function in Patients with Multiple Sclerosis. Smolders et al. 2009.
  11. Treating tumor-bearing mice with vitamin D 3 diminishes tumor-induced myelopoiesis and associated immunosuppression, and reduces tumor metastasis and recurrence. Young et al. 1995.
    READ ABSTRACT Email me for a PDF copy
  12. 1 α,25-Dihydroxyvitamin D3 activates T cells of tumor bearers through protein phosphatase 2A. Wiers et al. 1997.
    READ ABSTRACT Email me for a PDF copy
  13. Use of α,25-Dihydroxyvitamin D 3 treatment to stimulate immune infiltration into head and neck squamous cell carcinoma. Walsh et al. 2010.
  14. 1α,25-Dihydroxyvitamin D 3 to skew intratumoral levels of immune inhibitory CD34 + progenitor cells into dendritic cells. Kulbersh et al. 2009.
  15. Lessons from the bone marrow: how malignant glioma cells attract adult haematopoietic progenitor cells. Tabatabai et al. 2005.
  16. Dietary restriction reduces the incidence of 3-methylcholanthrene-induced tumors in mice: close correlation with its potentiating effect on host T cell functions. Konno et al. 1991.
    READ ABSTRACT Email me for a PDF copy
  17. PDE5 Inhibitors Enhance Tumor Permeability and Efficacy of Chemotherapy in a Rat Brain Tumor Model. Black et al. 2008.
  18. Phosphodiesterase-5 inhibition augments endogenous antitumor immunity by reducing myeloid-derived suppressor cell function. Serafini et al. 2006.
  19. Low-dose naltrexone suppresses ovarian cancer and exhibits enhanced inhibition in combination with cisplatin. Donahue et al. 2011.
  20. Synergistic effect of aged garlic extract and naltrexone on improving immune responses to experimentally induced fibrosarcoma tumor in BALB/c mice. Ebrahimpour et al. 2013.
  21. Opioid growth factor improves clinical benefit and survival in patients with advanced pancreatic cancer. Smith et al. 2010.
  22. Oral administration of Lactobacillus acidophilus induces IL-12 production in spleen cell culture of BALB/c mice bearing transplanted breast tumour. Yazdi et al. 2010.
  23. Lactobacillus casei ssp.casei Induced Th1 Cytokine Profile and Natural Killer Cells Activity in Invasive Ductal Carcinoma Bearing Mice. Dallal et al. 2012.
  24. Immune modulation and apoptosis induction: Two sides of antitumoural activity of a standardised herbal formulation of Withania somnifera. Malik et al. 2009.
    READ ABSTRACT Email me for a PDF copy
  25. A standardized root extract of Withania somnifera and its major constituent withanolide-A elicit humoral and cell-mediated immune responses by up regulation of Th1-dominant polarization in BALB/c mice. Malik et al. 2007.
    READ ABSTRACT Email me for a PDF copy
  26. Molecular insight into the immune up-regulatory properties of the leaf extract of Ashwagandha and identification of Th1 immunostimulatory chemical entity. Khan et al. 2009.
    READ ABSTRACT Email me for a PDF copy
  27. Effect of the alcoholic extract of Ashwagandha leaves and its components on proliferation, migration, and differentiation of glioblastoma cells: combinational approach for enhanced differentiation. Shah et al. 2009.
  28. Using the Heat-Shock Response To Discover Anticancer Compounds that Target Protein Homeostasis. Santagata et al. 2012.
  29. NF-kB-Induced IL-6 Ensures STAT3 Activation and Tumor Aggressiveness in Glioblastoma. McFarland et al. 2013.
  30. Effect of selenium on the immunocompetence of patients with head and neck cancer and on adoptive immunotherapy of early and established lesions. Kiremidjian-Schumacher, and Roy. 2001.
    READ ABSTRACT Email me for a PDF copy
  31. Cimetidine, an unexpected anti-tumor agent, and its potential for the treatment of glioblastoma (Review). Lefranc et al. 2006.
  32. Successful tumour immunotherapy with cimetidine in mice. Osband et al. 1981.
    READ ABSTRACT Email me for a PDF copy.
  33. Cimetidine reduction of tumour formation in mice. Gifford et al. 1981.
    READ SOURCE ABSTRACT Email me for a PDF copy.
  34. Cimetidine Activates Interleukin-12, Which Enhances Cellular Immunity. Ishikura, 1999.
  35. Cimetidine modulates the antigen presenting capacity of dendritic cells from colorectal cancer patients. Kubota et al. 2002.
  36. Perioperative cimetidine administration promotes peripheral blood lymphocytes and tumor infiltrating lymphocytes in patients with gastrointestinal cancer: Results of a randomized controlled clinical trial. Lin et al. 2004.
  37. Perioperative cimetidine administration improves systematic immune response and tumor infiltrating lymphocytes in patients with colorectal cancer. Li et al. 2013.
  38. Cimetidine suppresses lung tumor growth in mice through proapoptosis of myeloid-derived suppressor cells. Zheng et al. 2013.
    READ ABSTRACT Email me for a PDF copy.
  39. Combined cimetidine and temozolomide, compared with temozolomide alone: significant increases in survival in nude mice bearing U373 human glioblastoma multiforme orthotopic xenografts.
    READ ABSTRACT Email me for a PDF copy.
  40. Tadalafil reduces myeloid derived suppressor cells and regulatory T cells and promotes tumor immunity in patients with Head and Neck Squamous Cell Carcinoma. Weed et al. 2014.
    READ ABSTRACT Email me for a PDF copy.
  41. Tadalafil augments tumor specific immunity in patients with head and neck squamous cell carcinoma. Califano et al. 2015.
    READ ABSTRACT Email me for a PDF copy.
  42. Biological mechanism and clinical effect of protein-bound polysaccharide K (KRESTIN(®)): review of development and future perspectives. Maehara et al. 2012.
  43. Activation of antitumor immune responses by Ganoderma formosanum polysaccharides in tumor-bearing mice. Wang et al. 2014.
    READ ABSTRACT Email me for a PDF copy.
  44. Immune-mediated antitumor effect by type 2 diabetes drug, metformin. Eikawa et al. 2015.
    DOWNLOAD SOURCE DOCUMENT PDF plus Supporting Information
  45. COX-2 blockade suppresses gliomagenesis by inhibiting myeloid-derived suppressor cells. Fujita et al. 2011.
  46. Methionine enkephalin regulates microglia polarization and function. Xu et al. 2016.
    READ SOURCE DOCUMENT email me for a PDF copy


  1. I read recently that the maitake d-fraction used by Namba (which it alleged he isolated) is only available in those studies. That in the US/Canada maitake d-fraction is not the same thing, a company in the USA copyrighted the term maitake d-fraction but it doesn’t have the same properties that Nanba used. So all of the products that we see sold as maitake d-fraction (in the United States anyway) is not equivalent dosing. It may not have the d-fraction at all. I’ve been looking for info on how to contact Nanba to ask him directly. Have you come across his information, by any chance? It would be a shame if we’re taking major doses, and spending all that money, without results.

    • I plan on doing some serious investigation of the best available Maitake brand. The products labelled “Maitake D-fraction” that I’ve seen contain a small fraction of D-fraction, in the neighborhood of 5-10%. One brand (AOR) contains 240mg maitake TD fraction standardized for 10% D-fraction. Another brand, “Maitake D fraction Forte” contains 250mg maitake powder plus 50mg Maitake PD fraction (which is 30% D fraction), for a total of 5% D-fraction. Assuming that the “D-fraction” in these brands is indeed the D-fraction Nanba used, you would still have to consume 4 to 7 capsules a day to get the therapeutic 100mg dose. If anyone hears of a more concentrated source, please let us know. It would certainly be interesting to contact Nanba directly.

      • When I search Nanba and Maitake D-Fraction the first link I find is this:

        It is concerning because Nanba has his own line of mushroom supplements ( Why would he use Maitake products out of NJ. That link and article look frightfully fabricated – where’s the references? Either that or Maitake Products, Inc. is the same company as Maitake nutraceuticals.

        It’s tricky to know who to trust.

        • After digging a little, it seems that Nanba did have some connection to Maitake Products Inc, in New Jersey. The company has since changed its name to Mushroom Wisdom (East Rutherford, New Jersey) and has legal rights to the name “Maitake D-fraction”.

          Their two major D-fraction products are called Professional Strength and Standard Strength. Professional Strength is far more expensive. The major difference is that Standard Strength has 40 mg Maitake standardized extract per serving, while Professional Strength has 240 mg standardized extract per serving. PD-Fraction is listed in the ingredients. What is the difference between PD-Fraction and D-fraction? That is the next thing to find out.

  2. In the ingredients list, it states PD-fraction (30% active proteoglucan).

    Another brand, Nutrisan, has this in the ingredients “Maitake PD-Fraction (Grifola frondosa) extract
    standardized at 30% D-Fraction”

    I conclude that the “30% active proteoglucan” in PD fraction, is D-fraction.

    If this interpretation is correct, and 30% of the standardized extract is D-fraction, then you’d be getting 72mg D-fraction per 6 capsules of Professional Strength, and only 12mg D-fraction per 4 capsules of the Standard Strength product.

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