IMMUNOLOGICAL INVESTIGATIONS. 31(2).

137-153 (2002)

INCREASED TUMOR NECROSIS FACTOR ALPHA (TNF-<x) AND NATURAL KILLER CELL (NK) FUNCTION USING AN INTEGRATIVE APPROACH IN LATE STAGE CANCERS

ABSTRACT

Darryl See, * Stephanie Mason, and Ramesh Roshan

Center for Advanced Medicine, 4403 Manchester Ave. Suite 107, Encinitias, California 92024

Natural products may increase cytotoxic activity of Natural Killer Cells (NK) Tumor Necrosis Factor alpha (TNF-a) while decreasing DNA damage in patients with late-stage cancer. Pilot studies have suggested that a combination of Nutraceuticals can raise NK cell function and TNF-a alpha activity and result in improved clinical outcomes in patients with late stage cancer. The objective of the study is to determine if Nutraceuticals can significantly raise NK function and TNF levels in patients with late stage cancer. After informed consent was obtained, 20 patients with stage IV, end-stage cancer were evaluated (one bladder, five breast, two prostate, one neuroblastoma, two non-small cell lung, three colon, 1 mesothelioma, two lymphoma, one ovarian, one gastric, one osteosarcoma). Transfer Factor Plus (TFP", 3 tablets 3 times per day), IMUPlus' (non denatured milk whey protein, 40 gm/day); Intravenous (50 to 100 gm/day) and oral (1-2 gm/day) ascorbic acid; Agaricus Blazeii Murill teas (10 gm/day); Immune Modulator Mix (a combination of vitamin, minerals, antioxidants and immune-enhancing natural products); nitrogenated soy extract (high levels of genistein and dadzein) and Andrographis Paniculata (500 mg twice daily) were used. Baseline NK function by standard 4 h SICr release assay and TNF alpha and receptor levels were measured by ELISA from resting and phytohemagglutinin (PH A) stimulated adherent and non-adherent Peripheral Blood

*Corresponding author. Fax: 760-632-0574; E-mail: darrylsee.g cs.com

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Copyright (0 2002 by Marcel Dekker, Inc.

www.dekker.com

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Mononuclear Cell (PBMC). Total mercaptans and glutathione in plasma were taken and compared to levels measured 6 months later. Complete blood counts and chemistry panels were routinely monitored. As of a mean of 6 months, 16/20 patients were still alive. The 16 survivors had significantly higher NK function than baseline (p < .01 for each) and TNF-C( levels in all four cell populations studied (p < .01 for each). Total mercaptans (p < .01) and TNF-C( receptor levels were significantly reduced (p < .01). It was also observed that hemoglobin, hematocrit and glutathione levels were significantly elevated. The only toxicity noted was occasional diarrhea and nausea. The quality of life improved for all survivors by SF-36 form evaluation. An aggressive combination of immunoactive Nutraceuticals was effective in significantly increasing NK function, other immune parameters and hemoglobin from PBMC or plasma in patients with late stage cancers. Nutraceutical combinations may be effective in late stage cancers. Clinical outcomes evaluations are ongoing.

INTRODUCTION

Despite enormous expenditures, little real progress has been made in the treatment of most cancers. Cancer incidence in the United States has risen 60 percent since 1950 and 27 fold since 1900 according to Surveillance, Epidemiology, and End Results (SEER)_[lJ Each year one in two men and one in three women in the U.S. develop some form of cancer according to the American Cancer Society.U' Mortality has climbed dramatically. In 1999, 560,000 people died of cancer in the U.S. alone. The mortality rate for cancer in 1994 was 6 percent higher than in 1970. [IJ Major gains have been made using chemotherapy in childhood leukemias, testicular cancer and some rare cancers, but the ability of chemotherapy and radiation to alter mortality of most cancers is negligible at best. Because of these facts, many Americans are utilizing some sort of alternative medicine in their battle against cancer. Estimates range from 9% of patients by the National Cancer Institute (NCI) to 60% of patients by other estimates. It appears that only about 5% of patients completely abandon traditional treatments for altematives.F' There is a significant need for research to determine, which if any alternative therapies show promise in the treatment of cancers. The role of the immune system in fighting cancer was first proposed by Paul Ehrlich around 1900, and the concept renewed by Lewis Thomas in 1959 and MacFarlane Burnet in 1969. Various research centers have been looking at immunotherapy for some time, and in fact in certain cancers recombitant interleukin-2 (IL-2) and interferons are used, but these therapies are plagued with serious side-effects.P' Various natural products have been shown to have immune modulating properties.

The importance of the immune system in cancer prevention and adjuvant therapy has been well-established. Penn reports the

NK FUNCTION AND TFN-u PRODUCTION IN LATE STAGE CANCERS 139

following evidences for the role of the immune system in the development of cancers.[41

1. Children with immunodeficiency diseases have increased rates of lymphoma, leukemia and Hodgkin's disease.

2. High rates of Kaposi's sarcoma and lymphoma in HIV infected individuals.

3. In organ transplant patients on immunosuppressive medications, there is a 3 fold increase in malignancies including skin, lips, vulva, anus, liver, lymphoma and Kaposi's sarcoma.

4. Cancer risk increases with duration of immunosuppressive treatment. In a study of heart transplant patients, cancer incidence increased 3 fold after one year of immunosuppressive therapy and 26 fold after 5 y.

5. Patients with autoimmune diseases treated with immunosuppressive therapy showed increased incidence of acute leukemia, lymphoma, liver cancer, bladder cancer and skin cancer.

6. Secondary tumors are common in cancer patients who receive immunosuppressive chemotherapy treatment. These may include acute leukemias, lymphoma, and bladder cancers. However this increase may be due to the DNA damage caused by the chemotherapeutic agent.

The importance of Natural Killer (NK) cells in effective immunotherapy has been broadly accepted. Our laboratory has consistently found that stage 4 cancer patients typically have severely depressed NK function, and sometimes decreased numbers (unpublished data). This is consistent with a plethora of other research on the subjectPl Decreases in NK function are believed to be, at least in part, attributable to neoplasm's ability to "steer" the immune system towards T-helper 2 (Th2) vs. cell mediated or T-helper 1 (Thl) immunity. Many cancers secrete interleukin-IO (IL-IO) as part of this mechanism.

Human Tumor Necrosis Factor-alpha (TNF-rx) is a 233 amino acid residue, nonglycosylated polypeptide that exists as either a transmembrane or soluble protein. When expressed as a 26 kDa membrane bound protein, TNF-rx consists of a 29 amino acid residue cytoplasmic domain, a 28 amino acide residue transmembrane segment, and a 176 amino acid residue extracellular region. The soluble protein is a 17 kDa, 157 amino acid residue molecule that normally circulates as a homo trimer. The variety of cell types known to express TNF-alpha includes macrophages, CD4+ and CD8+ T cells adipocytes, keratinocytes, mammary and colon epithelium, osteoblasts mast cells, dendritic cells, pancreatic beta-cells, astrocytes, neurons, monocytes, and steroid-producing cells of the adrenal zona reticularis. [6] Results of adenoviral mediated gene transfer of human TNF -alpha indicate a reduced systemic toxicity with an enhanced local antitumor effect in mice_[71

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Gillio-Tos and colleagues demonstrated that TNF-alpha antitumoral effect was due to apoptosis mediated by the down-regulation of the antiapoptotic gene bc1-2_l8] Also, retroviral mediated gene transfer of the human TNFalpha (hTNF) gene into U373MG human glioblastoma cells was shown to result in a reduction of the cells growth rate as compared to the parental cells. [9]

Tumor necrosis factor has direct in vitro antitumor activity on 30 to 50% of cell lines. Because of its pleiotropy, it is not fully understood which mechanism is responsible for its necrotic and apoptotic activities. When membrane bound, TNF-alpha exhibits antineoplastic potential, but when free floating, is a negative prognosticator of survival, being associated with wasting both in cancer and AIDS patients.

Transfer factors are small peptides that act as immune system regulatory cytokines.i'Y' Transfer Factor Plus (TFpi() contains several immunoactive components that have been shown to act synergistically in raising NK function and also effective as adjuvant therapy in cancer therapy.l'f In fact, one study (submitted) has suggested that TFpE) alone may raise NK function by nearly 250%. One component, inositol hexaphosphate (IP-6), has been shown to have at least three anti-cancer properties (NK enhancer, induces p53 and diminishes mutagenesis). The other components, namely, three medicinal mushrooms, mannans, thymic lipoproteins, and beta glucan, have all been proven to enhance NK function as well as transfer factor itself.

Non-denatured milk whey protein isolates have been found to have a number of bioactive properties. Bovine milk contains around 3 % protein by weight, with only 6.3% of that protein being whey protein. Normal processes to separate the whey component from the other constituents leads to significant denaturing of the bioactive whey proteins. Non-denatured whey protein isolates utilize proprietary processes to attain a protein containing over 90% non-denatured whey protein. Bioactive components include lactoferrin, lysozyme, lactoperoxidase, glycomacropeptide, alpha-lactalbumin, and bovine serum albumin. Lactoferrin exhibits anticancer, antiviral, antibacterial, and antifungal activity. It plays an active role in iron transport, an active role in the cytotoxic defenses of neutrophils, and scavenges free iron which acts as a free radical.[12.13]

Of particular interest with non-denatured milk whey proteins, is their up-regulation of intracellular glutathione via supplying cystine. The roles of glutathione (GSH) has been summarized by Gutman[14] and include enhancement of immune function, elimination of toxins, elimination of carcinogens, antioxidant cell protection, protection from ionizing radiation, DNA synthesis and repair, protein synthesis, prostaglandin synthesis. leukotriene syntheses, amino acid transport, and enzyme activity and regulation. Bounous et al have demonstrated that non-denatured whey protein isolate formula increased lymphocyte intracellular glutathione by

NK FUNCTION AND TFN-G' PRODUCTION IN LATE STAGE CANCERS 141

Tubie I. Ingredients in Immune Enhancing Formula

Ingredient Amount

Co-enzyme QIO

Grape seed extract (95%) Alpha Carotene

Lipoic acid

Lycopene

Vitamin E (Mixed tocopherols) Vitamin C (Beet-source)

Folic acids

Vitamin A (Acetate) Vitamin B-1

Vitamin B-2

Vitamin B-3 (Niacinamide and Niacin) Pantothenic acid

L-Glycine

Vitamin B-6

Vitamin B-12 (Cyanocobalarnine) Biotin

IP-6 (Inositol hexaphosphate) Choline

Germanium sesquioxide N-Acetyl -L-Cysteine Glutathione L-Carnitine

Calcium (Ascorbate) Magnesium

Vitamin D-3

Zinc

Potassium Chromium Molybdenum Manganese Selenium

Iodine

MSM (Methylsulfonylmethane) Borage Oil (20% GLA) Tocotrienols

Bioflavenoid complex

TMG (Trimethyl Glycine) Sulforaphane (Broccoli)

Beta Glucan

Olive leaf extract

Milk thistle extract (80% Silymarin) Green tea extract (40% Catechines) Mushroom extract cordyceps sinensis,

Trernella fuciformis, Ganoderrna lucidum, Lentinula edodes, Grifola frondosa, Coriolus versicolor

Agaricus blazei

80mg 40 mg 5mg 15mg Img 66IU 1320mg 90 meg 3300IU 5mg 6mg 74mg

40mg 500mg 6mg 400mcg 55mcg 200mg 60mg

25mg IOOmg lSOmg 100mg

40mg 80mg 65IU IOmg 75mg

50mcg 25mcg 0.5mg 35mcg 2SI11cg IOOmg 150mg

25mg 40mg

200mg 45mg SOmg 80mg 2Smg 45mg

2200mg 200mg

(continued)

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Table I. Continued

Ingredient

Amount

Andrographis paniculata Bromelain

Turmuric extract (Curcumin 95%) Panax ginseng

Cats claw

L-Taurine

Lactoferrin

50mg l75mg 90mg 15mg l50mg IOOmg 60mg

greater than 120% in mice vs. mice fed standard, commercially available

. ., [1516J

whey protem concentrates, or casem proteins. .

Kennedy demonstrated that non-denatured whey protein isolate formula increased glutathione in normal cells, but decreased glutathione in cancer cells.[17] It has been proposed that cancer cells produce more intracellular glutathione than normal cells in order to protect themselves from various reactive oxygen species. Thus, it would appear possible that the ingestion of non-denatured whey protein isolates may oxidatively stress cancer cells, while protecting normal cells. Bounous et al. have also demonstrated that mice fed non-denature whey protein isolates exhibit a significantly smaller tumor load than controls, when both groups are fed known carcinogens.F'"

Additionally, wasting (cachexia) can be a significant problem for the cancer patient. Though the mechanisms of cachexia are not fully understood, it is clear that there are major metabolic alterations in the cancer patient.[19] Tumor cells usually resort to the anaerobic rather than the aerobic metabolism for the production of energy. This type of metabolism is grossly inefficient and generates large amounts of lactic acid that must in turn be regenerated back into glucose in the liver, via the cori cycle, which also requires additional amounts of energy. Eventually the body begins to consume skeletal muscle for energy. Cachexia is also characterized by decreased appetite. Non-denatured whey protein isolate supplies a concentrated form of easily digestible high efficiency protein, without the need to Consume large volumes of food. Finally, a number of clinics, including our own, have observed that patients rendered anemic due to chemotherapy and radiation appear to have robust recovery of their hemoglobin and hematocrit when fed adequate levels of non-denatured whey protein isolate (personal communication).

Our institute employs the use of a specialized powder containing a number of immune enhancing ingredients, including high potency antioxidants (Vitamins A, C, E, Selenium), products that enhance phase 2 detoxification in the liver, and products known to have effects on restoring cell cycle dynamics in cancer cells via a number of mechanisms including upregulation

NK FUNCTION AND TFN-O' PRODUCTION IN LATE STAGE CANCERS 143

Table II. Regime

1 Nutraceuticals:

• TFP'K, Imuf'lus" milk whey protein, Agaricus Blazeii Murill teas, Transfer Factor PlusE, Beta Glucan, immune augmenting powder, and Soy Extract (and others cancer specific e.g., PC-Spes for Prostate)

2 Hyperthermia-local and systemic (far infrared sauna) 3 Light generator

4 Filtered, magnetic resonant water 5 Enemas

6 Lymphatic massage 7 Immunoimagery

8 Vegetarian, low sugar diet 9 Digestive enzymes

10 DMSA

11 Intravenous semi-benzyl Ted ascorbic acid (50-10 grrr/day)

of tumor suppressor genes, downregulation of oncogenes, and other modulating effects on cyclin dependent kinases and other modulators of cell cycle dynamics (green tea extract, andrographis paniculata, Vitamin A, Vitamin D, to name a few)PO] The full regime each patient used is shown in Table II.

The purpose of this pilot study is to show if a regimen of natural immunomodulators could increase NK function and augment TNF-C( production from PBMC; decrease tumor load; and increase quality of life in end-stage patients with cancer.

MATERIALS AND METHODS

Patient Selection

Twenty sequential patients with previously known stage IV malignancies were selected to participate in this study at the Immune Institute. After informed consent was obtained, 20 patients with stage IV, end-stage cancer were enrolled (one bladder, five breast, two prostate, one neuroblastoma, two non-small cell lung, three colon, 1 mesothelioma, two lymphoma, one ovarian, one gastric, one osteosarcoma). Therapy and surgery within 1 month of the procedure were exclusion criteria. The protocol was explained to them and consent forms were signed as required by the local Institutional Review Board. Inclusion criteria included age 18 to 75; 1 month to 6 month survival diagnosed by a Board Certified Oncologist; palliative therapy only planned in the future and no evidence of acute organ failure. Exclusion criteria included patients with severe leukopenia; poor respiratory or renal function; heart failure above New York heart association grade II; ventricular arrhythmias; autoimmune disorders; and coagulation abnormalities (Table III).

144 SEE, MASON, AND ROSH AN

Table Ill. Patient Demographics

Average age Gender

Previous surgery Previous chemotherapy Previous radiation Stage

Liver involvement

A vg. length of diagnosis

Oncologist prognosis median survival Further standard therapy planned

49.3+/~I5.6

12 males, 8 females[20) 13

15

14

All stage IV 9

6.9 + / ~ 6.1 months 3.7 + / ~ 3.0 months 3/20 (all palliative)

Peripheral blood monocytic cells (PBMC) were isolated from the 400 mL of heparinized whole blood by density gradient centrifugation using Lymphocyte Separation Media (LSM), Cellgro' (Mediatech, Herndon, V A). The PBMC were washed three times with phosphate-buffered saline (PBS; Mediatech, Herndon, VA) to remove platelets and any traces of LSM. The washed cells were then used fresh for laboratory studies.

Laboratory Studies

NK Cell Activity

MOLT-4 cells (5 x 106, ATCC, Manassas VA) were incubated with 30 IlCi of 51-Chromium CS1Cr) for 1 h at 37°C with 5% CO2 The cells were washed once with PBS to remove excess chromium and transferred to a 96 well microplate. Each assay was done in triplicate and the results of the three wells averaged. Wells to assess total release contained 50 ul, of the labeled cells, 50 ul. of 3% triton-X and 100 ul, of RPM!. Wells to determine spontaneous release consisted of 50 ul, of labeled cells and 150 ul, of RPM!. Test wells contained 100 ul, of the PBMC (5 x 105/piate), 50 ul. (5000 cells) of the labeled MOLT-4 cells and 50 ul, of RPMI. The micro plate was then incubated for4h at 37°C with 5% CO2, Following the incubation, the plate was centrifuged at 4000 rpm for 15 min. A 100 ul, sample of the supernatant from each well was transferred into a test tube and radioactivity was counted for one minute with a gamma counter. A standard formula was used to calculate the NK activity.

Tumor Necrosis Factor-«

A dilution series for TNF-CI. standard was prepared as specified by the manufacturer's package insert (Sigma, St. Louis MO). 200 uL of each standard and samples were pipetted into the designated wells of a microplate. 50 ul, of assay diluent 1 F (PBS with 2% Fetal Bovine Serum [FBS]) was

NK FUNCTION AND TFN-O' PRODUCTION IN LATE STAGE CANCERS 145

added to each well. The plate was then incubated for 2 h at room temperature. Following the incubation, each well was washed three times with 400 ul. of washed buffer. Excess wash buffer was removed and 200 ul. of TNF-cx conjugate was pipetted into the wells. The plate was then incubated for 1 h at room temperature.

After the second incubation the wells are washed again. A 200 ~L of the substrate solution containing equal volumes of colored reagent A and B (kit component) was pipetted into the washed wells. The plate was then incubated for 20 min at room temperature. The reaction was stopped by adding 50 ~L of stop solution. The absorbance of each well was read using an ELISA micro plate reader set at 450 nm.

Soluble TNF Receptor Type I (sTNF RI)

A dilution series for human sTNF RI standard was prepared as specified by the manufacturer's package insert (R&D Systems, Minneapolis MN). 200 ~L of each standard and samples were pipetted into the designated wells of a 96-well microplate. 50 ~L of assay diluent RDIM was added to each well. The plate was then incubated for 2 h at room temperature. Following the incubation each well was washed three times with 400 ~L of washed buffer. Excess wash buffer was removed and 200 ~L of sTNF RI conjugate was pipetted into the wells. The plate was then incubated for 2 h at room temperature. After the second incubation the wells are washed again. A 200 ~L aliquot of the substrate solution containing equal volumes of colored reagent A and B was pipetted into the washed wells. The plate was then incubated for 20 min at room temperature. The reaction was stopped by adding 50 ~L of stop solution. The absorbance of each well was read using an ELISA microplate reader set at 450 nm.

Total Mercaptans

A dilution series for Total Mercaptan assay standards was prepared as specified by the manufacturer's kit (Calbiochem-Novabiochem, San Diego CA) package insert. 100 ~L of patient sample and standards was pipetted into spectrophotometer cuvettes. The total volume of the cuvettes containing the samples and standards was adjusted to 900 ~L with buffer solution #3. 50 ~L of solution RI (1 % trimethylbenzine [TMB]) was added to each cuvette mixed and the absorbance read at 356 nm using a spectrophotometer.

Glutathione

A dilution series for Glutathione assay standards was prepared as specified by the manufacturer's kit (Calbiochem-Novabiochern, San Diego CA)

146

SEE, MASON, AND ROSHAN

package insert. 1 00 ~L of patient sample and standards was pipetted into spectrophotometer cuvettes. The total volume of the cuvettes containing the samples and standards was adjusted to 900 JlL with buffer solution #3. 50 J..lL of solution RI was added to each cuvette mixed and the absorbance read at 356 nm using a spectrophotometer.

Hemaglobin, Hematocrit, Chemistry Panels

Standard complete blood count and chemistry panels were performed in the clinical laboratory by established methods.

STATISTICS

Differences in laboratory parameters between baseline and 4 weeks or 6 months were determined by ANOV A. Differences in projected vs. average survival were evaluated by chi square testing.

RESULTS

Twenty sequential late-stage cancer patients were that met inclusion criteria and were willing to participate were enrolled in order to avoid bias. By definition, the study inclusion criteria included an Oncologist projected survival of less than 6 months. Therefore, all patients technically should have been dead at 6 months. If one looks at the projected survival estimate (3.7 + / - 3.0), approximately 15 of the patients should be have been dead compared to only 4.

Immune parameters consistently and statistically improved over the 6 month tria1. NK function increased by over 400% (Table IV).

Four patients with very advanced cancer (mean estimated survival 1.5 months) died during the study. The patient with gastric cancer died after three weeks. The other three patients showed a partial response, dying between 4 and 6 months (bladder, non-small cell lung and colon). There was no correlation between type of tumor and response.

Many studies have verified the ability of NK cells to lyse cancer cells in ViVO_[15] Submitted data indicated that the synergistic, combination product TFP" alone could increase NK function by almost 250%. Combination nutraceuticals can be used to significantly boost NK function in the face of cancers that release toxins which inhibit NK function. The mean baseline NK function was 6.4 L U, which is significantly suppressed compared to normals. who have been determined to have over 20 LV activity.[5]

TNF is a cytokine that is a potent anticancer agent. Serum recordings of TNF can be deceiving because end-stage patients often have high circulating

NK FUNCTION AND TFN-a PRODUCTION IN LATE STAGE CANCERS 147
Table IV. Baseline and 6 Month Natural Killer Cell Function Results (LU)
Patient Baseline 4 Weeks 6 Months
3.7 18.6 20.4
2 1.2 14.8 26.8
3 5.5 34.6 21.3
4 6.6 32.7 29.3
5 11.7 36.6 36.1
6 10.4 29.2 32.6
7 7.4 33.6 30.1
8 8.8 28.2 19.6
9 4.8 21.5 24.3
10 10.1 31.2 33.2
11 6.5 32.4 31.1
12 2.4 21.3 22.8
13 5.3 28.9 25.6
14 7.8 21.6 33.7
15 1.8 11.6 20.8
16 6.6 15.6 34.6
Mean 6.4 25.7 27.6
> 400% P < 0.01 levels as their immune system attempts, but ultimately fails, to control metastatic cancers. High serum TNF levels have been associated with cachexia in end stage cancer patients due to its significant proinfiammatory properties.l'Y' However, evaluating the ability of a cancer patient's PBMC to release TNF alpha correlates with cancer killing and an ability to mount a response to the cancer.P' In the current study, PBMC-derived TNF alpha was increased by over 10,000% after 6 months on the regimen the subjects received (Table V). Baseline levels were all less than 40 pg/rnl., compared to an average person with approximately 300 to 600 pg/rnl, response under similar laboratory conditions.P'

TNF-alpha type I receptors are found in high numbers on tumor cells.

The significant decrease (p < .01; Table VI) found in this study could be considered a crude estimate of lowered tumor burden.

Likewise, circulating mercaptans are associated with damaged DNA, including carcinogenic genes. A significant decrease (p < .01; Table VII) in the current study could be viewed as a similar marker for lessened DNA damage, and perhaps lower concentrations of oncogenes.

Glutathione is a potent intracellular antioxidant that improves immune function and was shown to be significantly improved in the study. Of the supplements used, ImuPlus has been shown to be the greatest inducer of glutathione, and may have been primarily responsible for the systemic increase in the face of cancer, which typically lowers this protein.[13]

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Table V. Baseline and 6 Month Tumor Necrosis Factor Alpha Results (Adherent, Nonstimulated PBMC Subpopulation. pg/mL)

Patient Baseline 4 Weeks 6 Months
2.2 794.2 1238.1
2 2.6 914.4 1341.5
3 11.6 1014.8 827.1
4 4.3 981.6 993.5
5 10.1 1286.3 1003.1
6 18.8 1342.1 1254.2
7 3.7 871.6 737.2
8 12.7 900.6 1042.7
9 2.7 639.7 737.2
10 35.7 1264.6 1563.7
II 1.7 783.4 804.1
12 23.2 741.9 1768.3
13 3.6 339.5 1673.0
14 18.9 238.4 1163.9
15 19.9 1672. 7 3124.7
16 31.5 538.2 1327.4
Mean 12.4 895.0 1287.5
> 10.000% P<O.OI Table VI. Baseline and 6 Month Tumor Necrosis Factor Alpha Type I Receptor Results (Optical Density)

Patient Baseline 4 Weeks 6 Months
2.2 1.9 1.4
2 2.0 2.1 1.6
3 3.4 3.1 J.7
4 2.9 2.7 0.8
5 3.7 3.5 2.7
6 1.8 1.6 1.2
7 3.7 3.2 2.9
8 3.8 3.6 2.7
9 1.9 1.9 1.6
10 3.8 3.4 2.1
II 1.6 0.6 < 0.5
12 1.8 1.9 1.9
13 3.2 2.1 1.7
14 2.5 1.3 0.8
15 3.0 2.7 1.8
16 2.6 2.4 1.6
Mean 2.74 2.38 1.69
P<OOI NK FUNCTION AND TFN-cx PRODUCTION IN LATE STAGE CANCERS 149

Table VII. Baseline and 6 Month Serum Mercaptans Results (Optical Density)
Patient Baseline 4 Weeks 6 Months
2.6 2.4 2.0
:; 3.4 1.8 0.8
3 4.1 3.7 1.4
4 4.9 3.4 3.6
5 3.2 3.4 3.0
6 2.4 2.7 2.8
7 2.7 3.5 1.8
8 2.8 2.2 1.1
9 3.7 3.4 1.9
10 3.2 3.0 2.4
11 2.4 1.1 1.1
12 2.9 1.7 1.4
13 3.8 2.6 1.9
14 2.9 1.8 1.1
15 3.9 2.3 1.0
16 4.1 2.8 1.7
Mean 3.3 2.6 1.8
P<O.OI Table VIII. Plasma Glutathione and Hematocrit Levels (OD)
Glutathione Hematacrit
Patient Baseline 6 Months Baseline 6 Months
0.63 1.25 32.1 34.6
2 0.48 1.83 28.7 36.4
3 0.96 1.64 24.7 33.7
4 1.23 1.21 25.4 34.2
5 0.31 0.97 30.8 35.7
6 0.47 1.53 32.6 29.6
7 0.65 1.43 22.7 28.3
8 0.77 1.24 21.6 34.5
9 0.43 1.51 29.4 35.6
10 0.64 1.34 36.8 37.5
11 1.03 1.94 34.8 35.9
12 0.14 0.78 25.7 31.1
13 0.66 1.53 31.2 35.6
14 0.74 1.48 29.4 34.5
15 0.63 1.42 34.7 37.2
16 0.43 1.04 30.5 33.6
Mean 64 1.38 28.3 32.7
p< .01 p< .05 150

SEE, MASON, AND ROSH AN

The 16 survivors all had significantly improved quality of life by SF-36 evaluation at 6 months (p < 0.6). Therefore, not only was life extended, but the patients in general felt better on the current regimen.

Mean hemaglobin and hematocrit levels were significantly increased (Table VI), improving oxygen carrying capacity to tissues, a crucial defense to normal tissues in resisting an anaerobic environment conducive to cancer growth.lI8]

DISCUSSION

Adequate levels of vitamins, minerals, orthomolecular compounds such as vitamin C, and nutrition from food are essential for health, and is especially important during times of physical and emotional stress. Cancer is stressful to the body in many ways. Numerous biological pathways and enzymes require vitamins for proper functioning. The immune system is no exception. Critical nutrients on which the immune system depends include vitamin C, folic acid, beta-carotene, and the minerals manganese, selenium, and zinc, among many others.

The February 25th, 1994 issue of Science magazine reported that scientists had been able to extend the lifespan of fruit flies by 30% with supplementation with antioxidant vitamins such as C, E, A, beta-carotene and selenium. It is postulated that vitamins protect cells from cancer in a number of ways including strengthening the immune system (vitamins C, E, A, and beta-carotene and the minerals selenium, zinc and manganese), neutralizing carcinogens (C and E) and preventing DNA and cellular damage (vitamins A and E, beta carotene and the minerals selenium, zinc and manganese).

Ascorbic acid, when given at sufficiently high dosages, has demonstrated preferential cytotoxicity to tumor cells in vitro and in vivo. Levels required to be cytotoxic in vivo to tumor cells are not attainable via oral administration. Cytotoxic effect requires intravenous administration of 50 Gms or more.l21]

It is hypothesized that ascorbic acid exhibits cytotoxic activity via a pro oxidant effect. There is a 10 to 100 fold greater content of catalase in normal cells than in tumor cells. Due to this, cancer cells reach high levels of intracellular hydrogen peroxide leading to their destruction, while normal cells are protected.l22•23]

Andrographis Paniculata is an herb commonly used in Ayurvedic medicine which possesses a number of pharmacological properties. Of particular interest to us is the observation that the herb is involved in restoration of cell cycle dynamics in cancer cells, a phenomenon often referred to as dedifferentiationP'''

The medicinal mushroom Agaricus Blazei Murill, like other medicinal mushrooms, has been observed to possess significant ability to stimulate macrophages, increase natural killer cells, and have other immunomodulatory

;-.rK FUNCTION AND TFN-(X PRODUCTION IN LATE STAGE CANCERS 151

effects. It is believed that the various fractions of beta glucans in medicinal mushrooms are responsible for this effect, but it appears that different fractions possess different immunomodulatory properties.[25.26] More recently Takaku identified a substance (ergosterol) in Agaricus possessing antiangiogenic activityY6]

Glutathione is a potent intracellular tripeptide which vigorously binds damaging free radical molecules that would other wise harm the cell by several mechanisms. Non-denatured milk whey protein (ImuPlus) has been shown to be a potent inducer of glutathione, thereby reducing cellular damage and improving intracellular function. Interestingly, a study by Kennedy[17] showed that non-denatured milk whey protein increased glutathione in cancer cells.

Most patients with cancer usually have decreased hemaglobin and hematocrit levels. However, in unpublished, preliminary data, ImuPlus has been shown to increase these levels, improving oxygen carrying capacity to tissues, improving their resistance to cancer.

It is beyond the scope of the current publication to discuss the actions and ramifications of all the constituents of the regimen used by the 20 study subjects, but the combination accomplished the following: immune function was significantly increased; markers of tumor load such as total circulating mercaptans were decreased; life span was increased; quality of life was improved; and glutathione and hemoglobin were increased. Combination approaches such as that used in the study may be necessary to improve the dismal statistics in stage III and IV cancers by acting at several sites in cancer growth, gene expression, biochemistry and mechanisms of metastases.

ACKNOWLEDGMENTS

We are grateful for the technical assistance of Dr. Romin Roshan. We are appreciative of the free product and partial funding of the study provided by Swiss Bioceuticals, and a portion of TFP H provided free of charge by 4-1ife International.

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