IMMUNOLOGICAL INVESTIGATIONS, 31(2), 137±153 (2002)

INCREASED TUMOR NECROSIS FACTOR

ALPHA (TNF-a) AND NATURAL KILLER CELL
(NK) FUNCTION USING AN INTEGRATIVE
APPROACH IN LATE STAGE CANCERS

Darryl See,* Stephanie Mason, and Ramesh Roshan

Center for Advanced Medicine, 4403 Manchester Ave. Suite 107,

Encinitias, California 92024

ABSTRACT

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 signi®cantly 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, ®ve
breast, two prostate, one neuroblastoma, two non-small cell lung, three
colon, 1 mesothelioma, two lymphoma, one ovarian, one gastric, one
1
osteosarcoma). Transfer Factor Plus (TFP , 3 tablets 3 times per day),
1
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 pro-
ducts); 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 51Cr release assay and TNF alpha and recep-
tor levels were measured by ELISA from resting and phytohemagglu-
tinin (PHA) stimulated adherent and non-adherent Peripheral Blood

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

137

Copyright # 2002 by Marcel Dekker, Inc. www.dekker.com

138 SEE, MASON, AND ROSHAN

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
signi®cantly higher NK function than baseline (p < .01 for each) and
TNF-a levels in all four cell populations studied (p < .01 for each). Total
mercaptans (p < .01) and TNF-a receptor levels were signi®cantly
reduced (p < .01). It was also observed that hemoglobin, hematocrit and
glutathione levels were signi®cantly 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 e€ective in signi®cantly increasing NK
function, other immune parameters and hemoglobin from PBMC or
plasma in patients with late stage cancers. Nutraceutical combinations
may be e€ective 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).[1] 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.[1] 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.[1] 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 alternatives.[2] There is a signi®cant need for research to determine,
which if any alternative therapies show promise in the treatment of cancers.
The role of the immune system in ®ghting cancer was ®rst 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-e€ects.[3] 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-a PRODUCTION IN LATE STAGE CANCERS 139

following evidences for the role of the immune system in the development of
cancers.[4]
1. Children with immunode®ciency 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 treat-
ment. 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 e€ective 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 subject.[5] 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
(Th1) immunity. Many cancers secrete interleukin-10 (IL-10) as part of this
mechanism.
Human Tumor Necrosis Factor-alpha (TNF-a) 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-a 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 homotrimer. 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 e€ect in mice.[7]
140 SEE, MASON, AND ROSHAN

Gillio-Tos and colleagues demonstrated that TNF-alpha antitumoral e€ect

was due to apoptosis mediated by the down-regulation of the antiapoptotic
gene bcl-2.[8] Also, retroviral mediated gene transfer of the human TNF-
alpha (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 ¯oating, 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.[10] Transfer Factor Plus (TFP1) contains several
immunoactive components that have been shown to act synergistically in
raising NK function and also e€ective as adjuvant therapy in cancer
therapy.[11] In fact, one study (submitted) has suggested that TFP1 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
signi®cant 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-lactalbu-
min, 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-a PRODUCTION IN LATE STAGE CANCERS 141

Table I. Ingredients in Immune Enhancing Formula

Ingredient Amount

Co-enzyme Q10 80 mg
Grape seed extract (95%) 40 mg
Alpha Carotene 5 mg
Lipoic acid 15 mg
Lycopene 1 mg
Vitamin E (Mixed tocopherols) 66 IU
Vitamin C (Beet-source) 1320 mg
Folic acids 90 mcg
Vitamin A (Acetate) 3300 IU
Vitamin B-1 5 mg
Vitamin B-2 6 mg
Vitamin B-3 (Niacinamide and Niacin) 74 mg
Pantothenic acid 40 mg
L-Glycine 500 mg
Vitamin B-6 6 mg
Vitamin B-12 (Cyanocobalamine) 400 mcg
Biotin 55 mcg
IP-6 (Inositol hexaphosphate) 200 mg
Choline 60 mg
Germanium sesquioxide 25 mg
N-Acetyl -L-Cysteine 100 mg
Glutathione 150 mg
L-Carnitine 100 mg
Calcium (Ascorbate) 40 mg
Magnesium 80 mg
Vitamin D-3 65 IU
Zinc 10 mg
Potassium 75 mg
Chromium 50 mcg
Molybdenum 25 mcg
Manganese 0.5 mg
Selenium 35 mcg
Iodine 25 mcg
MSM (Methylsulfonylmethane) 100 mg
Borage Oil (20% GLA) 150 mg
Tocotrienols 25 mg
Bioflavenoid complex 40 mg
TMG (Trimethyl Glycine) 200 mg
Sulforaphane (Broccoli) 45 mg
Beta Glucan 50 mg
Olive leaf extract 80 mg
Milk thistle extract (80% Silymarin) 25 mg
Green tea extract (40% Catechines) 45 mg
Mushroom extract cordyceps sinensis,
Tremella fuciformis, Ganoderma lucidum,
Lentinula edodes, Grifola frondosa,
Coriolus versicolor 2200 mg
Agaricus blazei 200 mg

(continued)
142 SEE, MASON, AND ROSHAN

Table I. Continued

Ingredient Amount

Andrographis paniculata 50 mg
Bromelain 175 mg
Turmuric extract (Curcumin 95%) 90 mg
Panax ginseng 15 mg
Cats claw 150 mg
L-Taurine 100 mg
Lactoferrin 60 mg

greater than 120% in mice vs. mice fed standard, commercially available
whey protein concentrates, or casein proteins.[15,16]
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
signi®cantly smaller tumor load than controls, when both groups are fed
known carcinogens.[18]
Additionally, wasting (cachexia) can be a signi®cant 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
inecient 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 concen-
trated form of easily digestible high eciency 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 antiox-
idants (Vitamins A, C, E, Selenium), products that enhance phase 2 detoxi-
®cation in the liver, and products known to have e€ects on restoring cell cycle
dynamics in cancer cells via a number of mechanisms including upregulation
NK FUNCTION AND TFN-a PRODUCTION IN LATE STAGE CANCERS 143

Table II. Regime

1 Nutraceuticals:
 TFP1, ImuPlus1 milk whey protein, Agaricus Blazeii Murill teas, Transfer Factor
Plus1, 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 gm=day)

of tumor suppressor genes, downregulation of oncogenes, and other

modulating e€ects on cyclin dependent kinases and other modulators of cell
cycle dynamics (green tea extract, andrographis paniculata, Vitamin A,
Vitamin D, to name a few).[20] 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-a
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 malignan-

cies 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, ®ve 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 Certi®ed 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 ROSHAN

Table III. Patient Demographics

Average age 49.3 ‡ = 15.6

Gender 12 males, 8 females[20]
Previous surgery 13
Previous chemotherapy 15
Previous radiation 14
Stage All stage IV
Liver involvement 9
Avg. length of diagnosis 6.9 ‡ = 6.1 months
Oncologist prognosis median survival 3.7 ‡ = 3.0 months
Further standard therapy planned 3=20 (all palliative)

Peripheral blood monocytic cells (PBMC) were isolated from the

400 mL of heparinized whole blood by density gradient centrifugation using
1
Lymphocyte Separation Media (LSM), Cellgro (Mediatech, Herndon, VA).
The PBMC were washed three times with phosphate-bu€ered 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 (56106, ATCC, Manassas VA) were incubated with

30 mCi of 51-Chromium (51Cr) 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 mL of the labeled cells,
50 mL of 3% triton-X and 100 mL of RPMI. Wells to determine spontaneous
release consisted of 50 mL of labeled cells and 150 mL of RPMI. Test wells
contained 100 mL of the PBMC (56105=plate), 50 mL (5000 cells) of the labeled
MOLT-4 cells and 50 mL of RPMI. The micro plate was then incubated for 4 h
at 37 C with 5% CO2. Following the incubation, the plate was centrifuged at
4000 rpm for 15 min. A 100 mL 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

A dilution series for TNF-a standard was prepared as speci®ed by the

manufacturer's package insert (Sigma, St. Louis MO). 200 mL of each
standard and samples were pipetted into the designated wells of a microplate.
50 mL of assay diluent 1 F (PBS with 2% Fetal Bovine Serum [FBS]) was
NK FUNCTION AND TFN-a 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 mL of washed bu€er. Excess wash bu€er was removed and 200 mL of
TNF-a 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 mL 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 mL
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 R1)

A dilution series for human sTNF RI standard was prepared as

speci®ed by the manufacturer's package insert (R&D Systems, Minneapolis
MN). 200 mL of each standard and samples were pipetted into the designated
wells of a 96-well microplate. 50 mL of assay diluent RD1M 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 mL of
washed bu€er. Excess wash bu€er was removed and 200 mL 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 mL 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 mL 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

speci®ed by the manufacturer's kit (Calbiochem-Novabiochem, San Diego
CA) package insert. 100 mL 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 mL with bu€er solution #3. 50 mL
of solution R1 (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 speci-

®ed by the manufacturer's kit (Calbiochem-Novabiochem, San Diego CA)
146 SEE, MASON, AND ROSHAN

package insert. 100 mL 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 mL with bu€er solution #3. 50 mL
of solution R1 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

Di€erences in laboratory parameters between baseline and 4 weeks or 6

months were determined by ANOVA. Di€erences 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 de®nition, 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 trial. 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 veri®ed the ability of NK cells to lyse cancer cells in
vivo.[15] Submitted data indicated that the synergistic, combination product
1
TFP alone could increase NK function by almost 250%. Combination
nutraceuticals can be used to signi®cantly boost NK function in the face of
cancers that release toxins which inhibit NK function. The mean baseline NK
function was 6.4 LU, which is signi®cantly suppressed compared to normals,
who have been determined to have over 20 LU 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

1 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 signi®cant proin¯ammatory
properties.[19] 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.[6] 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=mL, compared to
an average person with approximately 300 to 600 pg=mL response under
similar laboratory conditions.[6]
TNF-alpha type I receptors are found in high numbers on tumor cells.
The signi®cant 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 signi®cant 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 signi®cantly 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]
148 SEE, MASON, AND ROSHAN

Table V. Baseline and 6 Month Tumor Necrosis Factor Alpha Results (Adherent, Non-
stimulated PBMC Subpopulation, pg=mL)

Patient Baseline 4 Weeks 6 Months

1 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
11 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 < 0.01

Table VI. Baseline and 6 Month Tumor Necrosis Factor Alpha Type I Receptor Results
(Optical Density)

Patient Baseline 4 Weeks 6 Months

1 2.2 1.9 1.4

2 2.0 2.1 1.6
3 3.4 3.1 1.7
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
11 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 < 0.01
NK FUNCTION AND TFN-a PRODUCTION IN LATE STAGE CANCERS 149

Table VII. Baseline and 6 Month Serum Mercaptans Results (Optical Density)

Patient Baseline 4 Weeks 6 Months

1 2.6 2.4 2.0

2 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 < 0.01

Table VIII. Plasma Glutathione and Hematocrit Levels (OD)

Glutathione Hematacrit

Patient Baseline 6 Months Baseline 6 Months

1 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 ROSHAN

The 16 survivors all had signi®cantly 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 signi®cantly increased
(Table VI), improving oxygen carrying capacity to tissues, a crucial defense
to normal tissues in resisting an anaerobic environment conducive to cancer
growth.[18]

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 ¯ies 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 suciently 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 e€ect requires intravenous administration of 50 Gms or more.[21]
It is hypothesized that ascorbic acid exhibits cytotoxic activity via a
prooxidant e€ect. 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.[22,23]
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 dedi€erentiation.[24]
The medicinal mushroom Agaricus Blazei Murill, like other medicinal
mushrooms, has been observed to possess signi®cant ability to stimulate
macrophages, increase natural killer cells, and have other immunomodulatory
NK FUNCTION AND TFN-a PRODUCTION IN LATE STAGE CANCERS 151

e€ects. It is believed that the various fractions of beta glucans in medicinal

mushrooms are responsible for this e€ect, but it appears that di€erent
fractions possess di€erent immunomodulatory properties.[25,26] More recently
Takaku identi®ed a substance (ergosterol) in Agaricus possessing anti-
angiogenic activity.[26]
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 rami®cations of all the constituents of the regimen used by the 20 study
subjects, but the combination accomplished the following: immune function
was signi®cantly 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
1
by Swiss Bioceuticals, and a portion of TFP provided free of charge by 4-life
International.

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