.

Chapter 42: Oxidative Stress ... Oxidative Stress in Cancer,
           AIDS, and Neurodegenerative Diseases
           edited by

           LUC MONTAGNIER
           Centre National de la Recherche Scientifique
           Institut Pasteur Paris, France

           RENE OLIVIER
           Institut Pasteur
           Paris, France

           CATHERINE PASQUIER
           Centre National de la Recherche Scientifique
           Faculte Xavier-Bichat
           INSERM U294
           Paris, France

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      Chapter 42
     Nutriceutical Modulation of Glutathione with a Humanized Native Milk
Serum Protein Isolate, IMMUNOCALTM: Application in AIDS and Cancer

Sylvain Baruchel and Ginette Viau
McGill University-Montreal Children's Hospital Research Institute,
Montreal, Quebec, Canada.
Rene Olivier Pasteur, Institute Paris, France
Gustavo Bounous  Montreal General Hospital, Montreal, Quebec, Canada
Mark A. Wainberg
Jewish General Hospital, Lady Davis Institute, Montreal, Quebec,
Canada
Sub-Topics:

Nutritional Immunomodulation and its relation to Glutathione synthesis
Function of HNMPI as a cysteine delivery system In vitro modulation of
intracellular Glutathione by ImmunocalTM In vitro anti-HIV and antiapoptotic
activity of HNMPI HNMPI supplementation in AIDS and wasting syndrome
Selective Glutathione modulation of breast cancer
cells and impact on cancer cell growth HNMPI in cancer clinical trials
Analogy between HNMPI ImmunocalTM and human milk
Conclusion
                         References
NUTRITIONAL IMMUNOMODULATION AND ITS RELATION TO GLUTATHIONE SYNTHESIS
Fresh, raw milk includes the group of proteins that remain soluble
in "milk serum." These proteins can be preserved in their native
form if extracted carefully from their natural source.
In 1981 it was discovered that normal mice fed a milk serum protein
concentrate (specially prepared under mild nondenaturing conditions)
exhibited a marked increase in the humoral immune response to a T
helper cell-dependent antigen (1). In the following years, numerous
experiments confirmed the consistency of this phenomenon (2-10).
Over a period of 12 years and based on these findings a humanized
native milk serum protein isolate (HNMPI) named ImmuocalTm was
developed (Immunotec Research Corporation Ltd., Montreal, Quebec,
Canada).
This property was found to be related, at least in part to a greater
production of splenic Glutathione (L-a-glutamylcysteinylglycine)
(GSH) during the oxygen-requiring antigen-driven clonal expansion of
the lymphocyte pool in animals fed with this bioactive HNMPI (9).
Adequate levels of GSH are necessary for lymphocyte proliferation in
the development of the immune response (11,12). Moderate but
sustained elevation of cellular GSH was also found in the liver and
the heart of healthy, old mice fed with this HNMPI for a prolonged
period. In addition, HNMPI markedly increased their life expectancy
in comparison to control animals fed nutritionally equivalent diets
(13). Glutathione is of major significance in cellular antioxidant
activity in what Meister called the "GSH antioxidant system" because
it participates directly in the destruction of reactive oxygen
compounds and also because it maintains in reduced form ascorbate
(vitamin C) and a-tocopherol (vitamin E), which also exerts an
antioxidant effect (14).

UNCTION OF HNMPI AS A CYSTEINE DELIVERY SYSTEM
What ingredient in IMMUNOCALTM makes it an effective "cysteine
delivery system"?  Systemic availability of oral GSH is negligible in man
(15) and there is no evidence for transport of GSH into cells (16). Thus, it
has to be synthesized intracellularly. This occurs in two steps: (a)
glutamylcysteine synthesis; (b) Glutathione synthesis. Even though
the inflow of cysteine, glutamate, and glycine might prove somewhat
limiting under selected circumstances, numerous observations have
shown that it is the transport of cysteine (or cystine, which usually is
promptly reduced to cysteine on cell entry) which tends to be the
rate-limiting event in GSH synthesis, whereas free cysteine does not
represent an ideal delivery system (17) because it is toxic and is
spontaneously oxidized. Cysteine present as the disulfide cystine released
during ingestion in the gastrointestinal tract is more stable than free
amino acid. GSH synthesis is  submitted to negative feedback inhibition by
the end-product GSH. The disulfide bond is pepsin- and trypsin-resistant,
but may be split by heat and mechanical stress (9). Cystine accounts for
about             90% of the low-molecular-mass cysteine in the blood
plasma, while             reduced cysteine is present only at extremely low
concentration             (18).
In a comparative study, we found that commercial milk serum
concentrates exhibiting far less bioactivity, including less GSH
promoting activity, contain about half the amount of serum albumin
(9) and 4 times less lactoferrin than HNMPI, expressed as percentage
of total milk serum protein. IMMUNOCALTM is produced in a
proprietary lenient process which results in the preservation of the
most thermolabile proteins in their native conformation. In the serum
albumin, there are 17 cystine residues per 66 kDa molecule and 6 GluCys
dipeptides (19); in lactoferrin there are 17 per 77kDa molecule and 4
Glu-Cys dipeptides (20); and in the @-lactalbumin there are 4 cystines in a
14,000 kDa molecule (19).
Table 1.

                       Molecular
                       Mass (kDa)ResiduesCysteine
                       residues
                       per moleculeCysteine (Cys)2
                       (disulfide)Glu-(Cys)2

                       b-Lactoglobulin18,400162520
                       a-Lactabumin14,200125840
                       Serum albumin66,00058235176
                       Lactoferrin77,00070840174

                       Source: Refs. 19, 20.
On the other hand, b-Lactoglobulin has only 2 cystines in a 18,4OO
kDa molecule (19), and IgG1, the predominant immunoglobulin in cow
whey, has only 4 disulfide bridges in a 166,OOO kDa molecule (Table
1). In addition, Meister and colleagues (16) have demonstrated that
the y-glutamylcysteine (Glu-Cys) precursors of GSH can easily enter
the cell and there be synthesized into GSH. It thus becomes
noteworthy that the most labile milk proteins-, serum albumin and
lactoferrin-are those which contain these putative GSH-promoting
peptide components.  Finally, the bioavailibility of the presumed active
component (cystine and Glu-Cys group) may be influenced by the coexistence
of the other proteins throughout the digestive-absorptive process. This
newly discovered property of HNMPI was found to be independent of its
nutritional value, as other proteins of similar nutritional
efficiency do not exhibit this unique property (1-10). The concept that a
specific biological activity can exist in addition to and independent of the
systemic effect of IMMUNOCALTm as a good protein source is further
substantiated by recent in vitro assays
(21).      The dietary provision of cystine is particularly relevant to the
immune system. The coordinated response of macrophages and
lymphocytes in the T cell-mediated immune response is regulated, in
part, by macrophage cystine uptake and subsequent release of reduced
cysteine into the local environment for uptake by lymphocytes. When
the antigen-presenting macrophages come into close contact with
antigen-specific T cells, they supply these cells with additional
amounts of cysteine and thereby raise their intracellular GSH level
(18).
The validity of this assumption is confirmed by the demonstration
that the immunoenhancing and GSH-promoting (data not shown) effect
of ImmunocalTm is abolished by buthionine suIfoximine, which
inhibits y-glutamylcysteine synthetase, the initial step in GSH
synthesis (17).

Figure 1. Incubation of PBMC for 72 h in the presence of
various amounts of ImmunocalTM. Each point represents the mean
±SD of 3 measurements of intracellular glutathione. *p<0.05.

Figure 2. Incubation of PBMC's for 72 h in the presence of
ImmunocalTM and other serum milk products: Percentage increase
in Glutathione.

IN VITRO MODULATION OF INTRACELLULAR GLUTATHIONE BY IMMUNOCALTM
We demonstrated that normal human lymphocytes cultured for 3 days
with HNMPI 100 µg/ml show an increase in intracellular GSH content
from 4.5 ± 0.4 to 10.5 ± 3.4 nmol/106 cells, p <0.01 (Figure 1).
This increase in GSH correlates with an increase in cellular
proliferation measured by thymidine incorporation (data not shown).
Table 2.   Presence of Cytopathic Effects in MT-4 Cells

                       TCID50/wella

                       IMMUNOCALTM
                       (µg/ml)2000200202
                    0++++++-
                       1++++++-
                       10++++-
                       100----
                       500----
                       1000----
                       a + Presence of cytopathic effects; - absence of
                       cytopathic effects.

Figure 3. ImmunocalTM has been shown to inhibit HIV
                 replication.
The increase in GSH is dose-dependent and has not been found for
casein or for any commercially available milk serum protein
concentrate (Figure 2).

IN VITRO ANTI-HIV and ANTIAPOPTOTIC ACTIVITY OF HNMPI
Clinically, there is direct evidence that HIV infection is associated with a
GSH deficiency in the peripheral blood mononuclear cells (PBMC) (18). The
depletion of intracellular GSH suggests an association between oxidative
stress and HIV infection. Oxidative stress may be one of the mechanisms that
contribute to disease progression and the wasting syndrome through mediators
of inflammation such as TNF-a. and IL-6. During this period of
progression, Glutathione is consumed owing to an increase in oxidative
stress.

Figure 4. Inhibition of early cell death by ImmunocalTM

GSH depletion, a consequence of chronic oxidative stress, is part of
the spectrum of HIV infection. GSH has, in addition, a crucial role
in lymphocyte function and cell survival.  IMMUNOCALTM functioning as a
cysteine delivery system can enhance GSH synthesis in vitro (Figure 1) and
inhibits HIV replication on a cord mononuclear cell system infected by HTL
V-IIIB (Figure 3).
IMMUNOCALTM also inhibits the formation of syncitium between
infected and noninfected cells. The inhibition of syncitium formation
occurred at the same concentration as inhibition of HIV replication (Table
2). This viral inhibition was not associated with any cytotoxicity.
IMMUNOCALTR, via its GSH-promoting activity, reduces apoptosis in
HIV-infected cells. Apoptosis was evaluated by flow cytometry on PBMC from
HIV-infected individuals (Dr. R. Olivier, AIDS and Retrovirus Department,
Pasteur Institute).
HIV-infected PBMC cultured at concentrations of IMMUNOCALTM of 100
ug/ml or higher were less prone to die of apoptosis than untreated
cells: 15% ± 2.6% vs. 37% ± 2.4, p<0.001 (Figure 4).

HNMPI SUPPLEMENTATION IN AIDS AND WASTING SYNDROME
Based on these pre-clinical data, we conducted a Canadian clinical
trial (Canadian HIV Trials Network) with IMMUNOCALTM in children
with AIDS and wasting syndrome. The major objective was to evaluate
 the effect of oral supplementation with IMMUNOCALTM on nutritional
parameters and intracellular blood lymphocyte GSH concentration in
children with AIDS and wasting syndrome. This was an open single-arm
pilot study of 6 months duration. Wasting syndrome and severe weight
loss within the 6 months preceding entry into the study was an
absolute criterion for entry.
IMMUNOCALTM was administered twice a day as a powder diluted in
water. In some patients, IMNUNOCALTM was administered via
nasogastric tube when necessary. The administered starting dose was
based on 20% of the total daily protein requirement and was
increased by 5% each month over 4 months to reach 35% of the total
protein intake at the end of the study. The total duration of the study was
6 months.  Weight, height, triceps skinfold and mid-arm muscle
circumferences,  CD4/CD8 counts, and peripheral lymphocyte GSH
concentrations (measured by spectrophotometric assay) were measured monthly.
Energy intake was assessed by the use of two independent 2-day food records
with , 2-3 week period between the food records. Each food record included a
weekday and a weekend, and the average of these records was calculated to
reflect the daily nutritional intake. Out of 14             patients
enrolled, 10 were evaluable. The ages of the patient were             from 8
months to 15 years. The 10 patients studied were enrolled in
four different centers across Canada: Montreal Children's Hospital
(Dr. S. Baruchel), The Hospital For Sick Children Toronto (Dr. S.
King), Children's Hospital for Eastern Ontario (Dr. U. Allen), and
Centre Hospitalier Laval Quebec (Dr. F. Boucher). Of the 4 remaining
patients, 2 lacked compliance after 2 months while the other 2 died of AIDS
progressive disease within the first 2 months of entry into  the study. None
of the deaths was related to the tested product.
None of the patients experienced any major toxicity such as diarrhea or
vomiting or manifestation of milk intolerance. One patient had to
stop IMMUNOCALTM transiently for minor digestive intolerance such as
nausea and vomiting (<twice/day) at month 3 and was subsequently
able to restart the treatment without any problem.
At the end of the study, all patients experienced a weight gain in
the range of 3.2% to 22% from their starting weight. The mean weight
gain for the group was 8.4% ± 5.7%. On analysis of the mean
percentage of requirement nutrient intake (RNI) per month for all
Table 3.   Changes from Baseline (expressed as percentage) at Weeks 24 and
36 in Weight, Anthropometric  Measurements and GSH in Patients Treated with
ImmunocalTM.

 Weight
 change%Mid-arm Muscle
circumference
change%Triceps
skinfold
change %PBMC GSH
change (%)

Patientwk 24wk 32wk 24wk 32wk 24wk 32wk 24wk 32
                      122.129.89.514.350.025.012.2-9.0
                       214.017.318.725.320.020.084.056.0
                       35.19.2-3.0-2.0-17.0-3.037.055.0
                       43.83.44.2NA-42.0NA305.0550.0
                       57.14.513.111.4-24.0-16.0-18.014.3
                       63.75.6-2.0-2.016.016.07.1174.0
                       72.5NA5.0NA-13.0NA54.2NA
                       814.218.2-3.12.041.043.017.362.4
                       98.97.9-4.0-8.0-30.0-39.0-6.650.9
                       107.0NA1.0NA41.0NA-1.6NA

The patients, no correlation was found between the weight gain and
any significant increase in the mean percentage of RNI, suggesting
reduced catabolism rather than an anabolic effect of IMMUNOCALTM.
Six of ten patients have demonstrated an improvement in their
anthropometric parameters such as triceps skinfold or mid-arm muscle
circumference independently of an increase in energy intake (Table 3).
Two groups of patients were identified in terms of GSH modulation:
responders and nonresponders. The responders were those who started
the study with a low GSH level.

Figure 5. Intracellular glutathione in HSC 4. Eash point
represents the mean ±SD of 3 measurements.   Up arrow [6]
indicates end of study.
Figure 6. Intracellular Glutathione in CHUL 1. Each point
represents the mean ± of three measurements.   Up arrow [7]
indicates end of study.

Figure 7. Intracellular Glutathione in MCH 3. Each point
represents the mean ± of three measurements.   Up arrow [6]
indicates end of study.

The nonresponders were those who started with a normal GSH level. A
positive correlation was found between increase in weight and
increase in GSH (Figures 5,6,7). No changes were found in terms of
blood lymphocyte CD4 cell count, but 2 patients exhibited an
increase in the percentage of their CD8 cells and 4 patients showed
a trend toward an increase in the number of NK cells.
In conclusion, this pilot study demonstrates that IMMUNOCALTM is
very well tolerated in children with AIDS and wasting syndrome and
is associated with an amelioration of the nutritional status of the
patient as reflected by weight and antrhopometric parameters.
Moreover, the GSH-promoting activity of IMMUNOCALTM in vivo seem to
be validated in 6 out of 10 patients. An international multicenter
double-blind randomized study is currently under way in France and
Canada in adults patients with AIDS and wasting syndrome.

SELECTIVE GLUTATHIONE MODULATION OF BREAST CANCER CELLS AND IMPACT ON CANCER
CELL GROWTH
The specific involvement of GSH in the carcinogenic process is
supported by the major role played by this compound in the detoxification of
carcinogens by conjugation (26). We demonstrated that feeding GSH-promoting
HNMPI to mice chronically treated with dimethylhydrazine (DMH) significantly
reduces the number and size of colon carcinomas induced by DMH (27,28).
These colon tumors appear to be similar to those found in the human insofar
as the type of lesions and the chemotherapeutic response characteristics are
concerned
(26). HNMPI feeding appears to exert an inhibitory effect not only on the
initiation (27) of cancer, but also on the progression of tumors
(28).  Recently, a direct inhibitory effect of HNMPI in human cancer cell
replication was confirmed (21,29,30). In other human cancer cell studies,
the inhibitory effect was found to be related to the serum albumin component
of milk serum (31) and most recently to @-lactalbumin (32). Feeding
lactoferrin to mice inhibited the growth of solid tumors and in addition
reduced lung colonization by melanomas (33). Unlike other proteins, serum
albumin was found to exhibit a strong antimutagenic effect in an in vitro
assay using hamster cells (34). It is therefore noteworthy that in this
HNMPI we have succeeded in concentrating serum albumin, @-lactalbumin, and
lactoferrin, all containing a significant number of GSH precursors.
A possible explanation for these newly discovered properties of dietary milk
serum protein may be found in recent findings on the role of GSH in tumor
biology (35).
The search for ways to inhibit cancer cells without injuring normal
cells has been based over the years on a vain effort to identify the
metabolic parameters in which cancer cells are at variance with normal
cells. One such function could well be the all-important synthesis of
cellular GSH.  Recent experimental evidence has revealed an intriguing
response of tumor versus normal cells to GSH synthesis-promoting compounds.
Cellular GSH levels have been found to be several times higher in human
cancer cells than in adjacent normal cells (35). This finding is presumably
related to their proliferative activity. In fact, cancer is the only
condition in which elevation of such a tightly regulated system as GSH has
been reported. However, when a cysteine- and GSH-promoting compound such as
2-l-oxothiazolidine-4-carboxylate (OTZ) was added to cultured human lung
cancer cells exhibiting very high levels of GSH at the outset, no
intracellular increase was noted, whereas GSH increased substantially in
normal cells (35). This differential response is even more pronounced in
vivo. We demonstrated that in tumor-bearing rats, OTZ treatment was actually
found to deplete GSH in the tumors (36).

Figure 8. Intracellular Glutathione in MATB WT. Each point
                 represents the mean ±SE of three measurements. *p<0.05.

More specifically, an in vitro assay showed that, at concentrations that
induce GSH synthesis and proliferation in normal human cells
(Figure 1), IMMUNOCALTM caused GSH depletion and inhibition of
proliferation of cells in a rat mammary carcinoma (Figure 8) and
Jurkat T cells (Figure 9) (21). The selectivity demonstrated in these
experiments may be explained by the fact that GSH synthesis is negatively
inhibited by its own synthesis and since, as mentioned, baseline
intracellular GSH in tumor cells is much higher than in normal cells, it is
easier to reach the level at which negative feedback inhibition occurs in
this cellular system than in a nontumor cellular system.

Figure 9.   Intracellular Glutathione in JURKAT. Each point
represents the mean ±SE of three measurements.

HNMPI IN CANCER CLINICAL TRIALS
On the basis of these experiments, 5 patients with metastatic
carcinoma of the breast, 1 of the pancreas, and 1 of the liver, were
fed 30 g of IMMUNOCALTM daily for 6 months. In 6 patients, the blood
lymphocyte GSH levels were substantially above normal at the outset,
probably reflecting high tumor GSH levels. At completion of the 6
months of daily supplementation, 2 patients exhibited signs of tumor
regression, normalization of hemoglobin and peripheral lymphocytes
counts, and a sustained drop of lymphocyte GSH levels toward normal.
Two patients showed stabilization of the tumor and increases in
hemoglobin levels. In 3 patients, the disease progressed with a trend toward
higher lymphocytes GSH levels (37).  A major problem in the use of
chemotherapeutic agents in cancer therapy is the protection offered by the
defense mechanisms of cancer cells. An important element of protection is
represented by GSH, which is an effective detoxification agent that is
relatively abundant in tumor cells. Indeed, when GSH synthesis is inhibited
by buthionine sulfoximine (BSO), the activity of several
chemotherapeutic agents such as alkylating agents is increased and
drug resistance can be reversed (36-38). However, the concomitant
depletion of GSH in normal cells greatly limits the practical usefulness of
this modality of treatment.
We recently demonstrated that a selective GSH prodrug such as OTZ
protects some normal tissue (36) but also potentiates the activity of some
alkylating agents (38). The apparently selective depletion of tumor GSH
levels by provision of a natural precursor of GSH as contained in
IMUNNOCALTm seems to be associated with inhibition of proliferation of
cancer cells in vitro. This natural precursor of GSH favorably influences
the GSH synthesis in normal cells. These in vitro and preliminary clinical
results indicate that this newly discovered property of HNMPI may be a
promising adjunct to the nutritional management of cancer patients
undergoing chemotherapy.
We are currently developing a phase II study in breast carcinoma,
attempting to confirm that this selective depletion of GSH may, in
fact, render tumor cells more vulnerable to chemotherapy and
eventually protect normal tissue against the deleterious effect of
chemotherapy.

ANALOGY BETWEEN HNMPI IMMUNOCALTM AND HUMAN MILK            Human milk
contain about 80% of whey protein and 20% of casein. The
opposite is true for cow milk. An analysis of the mass ratio of casein to
whey protein in milk from various mammals clearly indicates that human milk
has the lowest ratio in any mammalian species (39). On the basis of our
laboratory studies showing the immunoprotective and anticancer effects of
cow whey protein concentrate, it is tempting to speculate that this
predominance of whey proteins in human milk is advantageous and thus
represent an evolutionary adaptation.

Scientific data based on the similarity between the bioactive
components of this native milk protein isolate (HNMPI) of cow milk,
IMMUNOCAL, and human whey protein appear to substantiate this
theory, as will now be discussed in more detail. It is well known that
breast feeding is superior to the use of cow milk-based formulas of similar
nutritional efficiency for the health of human babies. Breast feeding
protect against otitis media, and  pneumonia (40,41). Mothers milk also has
a protective effect on the incidence of several types of childhood cancer
including leukemia,  lymphomas, bone tumors, and brain tumors (42). Children
who are artificially fed or are breast fed for only a shot period of time
are more at risk for developing several types of cancer before the
age of 15 years as compared to long-term breast feeders (43). Thus,
the concept of a biological activity in addition to but independent of the
nutritional efficiency, formulated to describe the immunoenhancing and
GSH-promoting activity of the HNMPI IMMUNOCALTm,  may indeed apply to the
breast feeding of neonates and infants.  Glutathione synthesis appears to be
the crucial factor in the health benefit of HNMPI.
It may then be appropriate to identify the features common to HNMPI
and human whey proteins that are capable of influencing GSH
synthesis in the host. Cysteine, a crucial limiting factor in the
synthesis of GSH, is about as abundant in cow's whey protein as it is in
whole human milk proteins and several times more abundant than
in cow's whole milk (39), since most caseins contain either no
cysteine or one or two cysteine residues(19). As mentioned earlier,
our studies showed that the most thermolabile milk proteins, namely,
serum albumin, a-lactalbumin, and lactoferrin, are crucial to
expression of the bioactivity of HNMPI. As shown in Table 1, these
proteins are rich in cystine and glutamylcystine residues, natural
precursors of GSH. The presence of these dipeptides in the product
IMMUNOCALTM is a characteristic shared with human milk (Table 4).
Traditionally, it has been advocated that "humanized" cow milk
should contain more a-lactalbumin because this protein is twice as
abundant in human milk. On the basis of our experimental findings,
we propose instead that the principal health factor in human milk,
Table 4.   Protein Composition of Cow and Human Milk
                       Composition (g/liter)

                       ComponentCow milkHuman milk

                       ( 0 or 2
                       cysteine/molecule
                       no disulfide bond )
                       Casein (g/l)263.2
                       b-Lactoglobulin(g/l)3.2Neglible
                       a-Lactabumin (g/l)1.22.8
                       Serum albumin (g/l)0.40.6
                       Lactoferrin (g/l)0.142.0
                       Total cystine (mol/L)8.19 x 10-413.87 x 10-4
                       Total Cystine (mg/g protein)6.438.7

                       Source: Ref.19; Jennes R. Inter-species comparison
of
                       milk proteins. In fox, ed. Developments in dairy
                       chemistry-1. New York: ASP;1982:8
not denatured by heat pasteurization, is due to the predominance of the
thermolabile proteins rich in cystine and containing the Glu-Cys dipeptide
which are characteristic of the bioactive HNMPI, namely, serum albumin,
a-lactalbumin, and lactoferrin. This HNMPI differs from other commercially
available milk serum protein concentrates in having a relatively high
content of serum albumin (about 10%), lactoferrin (about 0.65 %), and
a-lactalbumen (about 28%). The variety of diseases against which breast
feeding appears to be effective suggest a broader protective mechanism
involving cellular GSH and its effect on free radicals, lymphocyte
proliferation, and detoxification of carcinogens and other xenobiotics.

 CONCLUSION
The biological activity of the proteins isolated from cow's milk in
ImmunocalTM depends on the preservation of those labile proteins
which share with the predominant human milk proteins the same
extremely rare GSH-promoting components. Cellular GSH depletion has
been implicated in the pathogeneses of a number of degenerative
conditions and disease states including Parkinson's, Alzheimer's,
arteriosclerosis, cataracts, cystic fibrosis, malnutrition, aging,
AIDS, and cancer (9).
This newly discovered nutriceutical modulation of GSH by the use of
humanized native milk serum protein isolate of bovine origin in AIDS
and cancer may well find other applications in disease where
oxidative stress and pathology of GSH metabolism are largely
implicated. Extensive pharmacoepidemiological study of GSH
metabolism and standardized methods of measurement of intracellular
GSH applicable in clinical trials are needed in order to better define the
clinical application of this new type of therapy.

            REFERENCES
             Bounous G, Stevenson MM, Kongshavn PAL. Influence of dietary
             lactalbumin hydrolysate on the immune system of mice and
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1: Anticancer Res. 1995 Nov-Dec;15(6B):2643-9. Related Articles, Links

The use of a whey protein concentrate in the treatment of patients with
metastatic carcinoma: a phase I-II clinical study.

Kennedy RS, Konok GP, Bounous G, Baruchel S, Lee TD.

Department of Surgery, Dalhousie University, Halifax, Nova Scotia, Canada.

Glutathione (GSH) concentration is high in most tumour cells and this may be
an important factor in resistance to chemotherapy. Previous in-vitro and
animal experiments have shown a differential response of tumour versus
normal cells to various cysteine delivery systems. More specifically, an
in-vitro assay showed that at concentrations that induce GSH synthesis in
normal human cells, a specially prepared whey protein concentrate,
Immunocal, caused GSH depletion and inhibition of proliferation in human
breast cancer cells. On the basis of this information five patients with
metastatic carcinoma of the breast, one of the pancreas and one of the liver
were fed 30 grams of this whey protein concentrate daily for six months. In
six patients the blood lymphocyte GSH levels were substantially above normal
at the outset, reflecting high tumour GSH levels. Two patients (#1, #3)
exhibited signs of tumour regression, normalization of haemoglobin and
peripheral lymphocyte counts and a sustained drop of lymphocyte GSH levels
towards normal. Two patients (#2, #7) showed stabilisation of the tumour,
increased haemoglobin levels. In three patients (#4, #5, #6,) the disease
progressed with a trend toward higher lymphocyte GSH levels. These results
indicate that whey protein concentrate might deplete tumour cells of GSH and
render them more vulnerable to chemotherapy.

Publication Types:
Case Reports
Clinical Trial
Clinical Trial, Phase I
Clinical Trial, Phase II

PMID: 8669840 [PubMed - indexed for MEDLINE]

Anticancer Res. 1996 May-Jun;16(3A):1095-9. Related Articles, Links

In vitro selective modulation of cellular glutathione by a humanized native
milk protein isolate in normal cells and rat mammary carcinoma model.

Baruchel S, Viau G.

Department of Pediatrics and Oncology, McGill University, Montreal, Quebec,
Canada.

We report the in vitro selective inhibitory activity of a humanized whey
protein concentrate IMMUNOCAL on growth of mammary carcinoma cells and
Jurkat T cells in comparison to normal peripheral blood mononuclear cells.
We relate this inhibitory activity to a selective depletion of intracellular
glutathione synthesis. The use of humanized whey protein concentrate as a
food supplementation may have direct implication in clinical trial with
adjuvant chemotherapy.

PMID: 8702219 [PubMed - indexed for MEDLINE]

1: Nutr Cancer. 2000;38(2):200-8. Related Articles, Links

Enchancing effect of patented whey protein isolate (Immunocal) on
cytotoxicity of an anticancer drug.

Tsai WY, Chang WH, Chen CH, Lu FJ.

Department of Biochemistry, College of Medicine National Taiwan University,
Taipei, ROC.

To determine the enhancing effect of a whey protein isolate on the
cytotoxicity of a potential anticancer drug, baicalein, the human hepatoma
cell line Hep G2 was assigned to grow in different media for four days, and
cell growth and apoptosis were investigated. The control group was grown in
normal medium; the other three groups were grown in whey protein isolate
(Immunocal) medium, baicalein medium, and a combination of Immunocal and
baicalein. As indicated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide assay, survival rate was significantly lower in cells
grown in baicalein + Immunocal than in cells grown in baicalein alone. In
contrast, there was no significant difference in survival rate of the cells
grown in Immunocal. In the investigation of apoptosis, cells grown in
baicalein + Immunocal showed a higher phosphatidylserine exposure, lower
mitochondrial transmembrane potential, and nearly 13 times more cells
undergoing apoptosis than cells grown in baicalein alone. We also
demonstrated that Immunocal reduced glutathione (GSH) in Hep G2 cells by
20-40% and regulated the elevation of GSH, which was in response to
baicalein. In conclusion, Immunocal seemed to enhance the cytotoxicity of
baicalein by inducing more apoptosis; this increase in apoptotic cells may
be associated with the depletion of GSH in Hep G2 cells. This is the first
study to demonstrate, in vitro, that Immunocal may function as an adjuvant
in cancer treatments.

PMID: 11525598 [PubMed - indexed for MEDLINE]

1: Nutr Cancer. 2000;38(2):200-8. Related Articles, Links

Enchancing effect of patented whey protein isolate (Immunocal) on
cytotoxicity of an anticancer drug.

Tsai WY, Chang WH, Chen CH, Lu FJ.

Department of Biochemistry, College of Medicine National Taiwan University,
Taipei, ROC.

To determine the enhancing effect of a whey protein isolate on the
cytotoxicity of a potential anticancer drug, baicalein, the human hepatoma
cell line Hep G2 was assigned to grow in different media for four days, and
cell growth and apoptosis were investigated. The control group was grown in
normal medium; the other three groups were grown in whey protein isolate
(Immunocal) medium, baicalein medium, and a combination of Immunocal and
baicalein. As indicated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide assay, survival rate was significantly lower in cells
grown in baicalein + Immunocal than in cells grown in baicalein alone. In
contrast, there was no significant difference in survival rate of the cells
grown in Immunocal. In the investigation of apoptosis, cells grown in
baicalein + Immunocal showed a higher phosphatidylserine exposure, lower
mitochondrial transmembrane potential, and nearly 13 times more cells
undergoing apoptosis than cells grown in baicalein alone. We also
demonstrated that Immunocal reduced glutathione (GSH) in Hep G2 cells by
20-40% and regulated the elevation of GSH, which was in response to
baicalein. In conclusion, Immunocal seemed to enhance the cytotoxicity of
baicalein by inducing more apoptosis; this increase in apoptotic cells may
be associated with the depletion of GSH in Hep G2 cells. This is the first
study to demonstrate, in vitro, that Immunocal may function as an adjuvant
in cancer treatments.

PMID: 11525598 [PubMed - indexed for MEDLINE]

1: Anticancer Res. 2003 Mar-Apr;23(2B):1411-5. Related Articles, Links

The antioxidant system.

Bounous G, Molson JH.

Research and Development Department, Immunotec Research Ltd., 292 Adrien
Patenaude, Vaudreuil-Dorion, Quebec, Canada J7V 5V5.

The glutathione (GSH) antioxidant system is the principal protective
mechanism of the cell and is a crucial factor in the development of the
immune response by the immune cells. Experimental data demonstrate that a
cysteine-rich whey protein concentrate represents an effective cysteine
delivery system for GSH replenishment during the immune response. Animal
experiments showed that the concentrates of whey protein also exhibit
anticancer activity. They do this via the GSH pathway, the induction of p53
protein in transformed cells and inhibition of neoangiogenesis.

Publication Types:
Review

PMID: 12820403 [PubMed - indexed for MEDLINE]

1: Anticancer Res. 2004 Mar-Apr;24(2B):553-4. Related Articles, Links

Molecular pathogenesis and prevention of prostate cancer.

Bounous G, Beer D.

Research and Development Department, Immunotec Research Ltd.,
Vaudreuil-Dorion, Quebec, Canada. jmolson@immunotec.com

Studies in laboratory animals indicate inhibition of chemically-induced
carcinoma by cystine-rich diets enhancing the cysteine-GSH antioxidant
system. The progression of carcinoma of the prostate is also inhibited by
these diets, which were later found to raise the level of GSH in the
prostate epithelium of man. New data presented at the July 13, 2003 meeting
of the American Association for Cancer Research indicates that higher levels
of total cysteine in plasma may predict a reduced risk for breast cancer.
This prospective investigation was conducted among 32,000 women in the
Nurses Health study. The previously reported prostate cancer data appears
then not to be strictly gender-related as the antioxidant role of the
cysteine-GSH system may also apply to breast cancer prevention.

Publication Types:
Review

PMID: 15160993 [PubMed - indexed for MEDLINE]

maybe if you read the doctor's book, you find your evidence. it seems
logical to me