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Paolo Lissoni1,*, Giusy Messina1,
Fernando Brivio2, Luca Fumagalli2, Luigi
Vigoré3, Franco Rovelli3, Luisa Maruelli4,
Mauro Miceli4, Paolo Marchiori4, Giorgio
Porro1, Michael Held5, Giuseppe di Fede6,
Toshi Uchiyamada7
Summary
In the last years, several immunomodulating antitumor
agents have demonstrated in the nature, particularly from Aloe
plant and rice bran. However, the major problem concerning the
natural antitumor agents is to define their immune mechanisms of
action in relation to the more recent advances in tumor
immunobiology. At present, the main cause responsible for the
lack of an effective antitumor response in advanced cancer
patients is belived to be represented by the generation of a
subtype of T helper lymphocytes (CD4+) with suppressive activity
on anticancer immunity, the so-called T regulatory lymphocytes
(T reg), which may be clinically identified as CD4+CD25+ cells.
On this basis, a study was planned to evaluate the effect of
rice bran extract arabinoxylan on T reg cell count and
percentage in solid tumor patients in relation to the various
lymphocyte subpopulations. The study included 22 evaluable
cancer patients, 16 of whom had an untreatable metastatic solid
tumor. Arabinoxylan was given orally at a dose of 2000 mg/day
for the first month, followed by a dose of 1000 mg/day for the
next month. In each patient we evaluated by monoclonal
antibodies the absolute number of lymphocytes, T lymphocytes
(CD3+), T helper (TH) lymphocytes (CD4+), T cytotoxic
lymphocytes (CD8+), NK cells (CD16+CD56+), T reg lymphocytes
(CD4+CD25+) and TH/T reg ratio before and after 2 months of
therapy. No substantial change occurred on therapy in the mean
number of lymphocytes, CD3+, CD8+ and NK cells. On the other
hand, the mean number of TH cells increased, whereas that of T
reg cell decreased on treatment, even though none of these
differences was statistically significant. On the contrary, TH/T
reg mean ratio significantly enhanced after arabinoxylan therapy.
In addition to its previously demonstrated stimulatory action on
NK function, this study shows that arabinoxylan may inhibit the
production of T reg cells, which are responsible for
cancer-related immunosuppression, with a following improvement
in the anticancer immunity. If further studies will confirm
these results, arabinoxylan could be successfully associated
with chemotherapy to induce not only a cytotoxic destruction of
cancer cells, but also an improvement in the immune status.
I. Introduction
The recent advances in the definition of the mechanisms
responsible for tumor progression have suggested the possibility
to control cancer growth not only trough chemotherapy-induced
cancer cell destruction, but also by stimulating the anticancer
immunity. In addiction to the exisence of endogenous antitumor
molecules, several agents capable of stimulating the anticancer
immunity have alsso isolated from plants. However, the
immunomodulatory effects of most natural immunomodulating agents
need to be better investigated in an attempt to establish their
mechanisms of action in relation to the most recent discoveries
concerning the physiopathology of the anticancer immunity. At
present, Aloe extracts (Lissoni et al,
1998) and arabinoxylan extract from rice bran (Ghoneum
and Jewett, 2000) would represent some of the potential
natural agents which could be utilized in the complementary
therapy of human neoplasms. Today, it is known that the
antitumor immune response is the end-result of several
interactions involving cytokines and immune cells, provided by
stimulatory or suppressive effects on the anticancer immunity (Atzpodien
and Kirchner, 1990; Rosenberg, 1992). Therefore, the lack
of an effective anticancer immune response in most cancer
patients with advanced disease would simply depend on the
prevalence of immunosuppressive mechansisms with respect to the
immunostimulatory ones (Atzpodien and
Kirchner, 1990). The anticancer immunity is mainly
activated by T helper-type 1 lymphocytes by releasing IL-2 (Whittington
and Faulds, 1993), and by dentritic cells, which act as
antigen-presenting cells producing IL-12 (Banks
et al, 1995), T cytotoxic lymphocytes and NK-LAK system,
which are involved in the induction of the antigen-dependent and
antigen-independent cytotoxicity, respectively (Atzpodien
and Kirchner, 1990). Therefore, IL-2 and IL-12 would
represent the main anticancer cytokines in humans. On the
contrary, the suppression of the anticancer immune response is
mediated by several cytokines, namely IL-10 (Moore
et al, 1993), IL-6 (Matsuda and
Hirano, 1990) and TGF-β (Shevach,
2002). Recently, however, it has been demonstrated that
the various endogenous suppressive factors would exert their
inhibitory immune effect through a common end-mechanism,
consisting of the generation of a subtype of T helper
lymphocytes (CD4+cells), provided by a fundamental suppressive
activity on the anticancer immunity, the so-called T regulatory
lymphocyte (T reg) (Dieckmann et al, 2001),
which at present seems to constitute the main mechanism
responsible for cancer-related immunosuppressive status. T reg
cells may be identified by the simultaneous expression of the
alpha-chain of IL-2 receptor (CD25) and CD4 antigen (Dieckmann
et al, 2001). Then, T reg cells may be clinically
recognized as CD4+CD25+ lymphocytes. Therefore, each eventual
natural immunomodulating agent would have to be investigated in
relation to its possible effect on T reg generation since, at
least from a theoretical point of view, each natural agent
capable of counteracting T reg activity could positively
influence the prognosis of the neoplastic disease by improving
the efficacy of the anticancer immune response. Moreover, our
previous preliminary studies have suggested that the percentage
of T reg cells with respect to the total number of T helper
cells, as expressed as CD4/CD4CD25 ratio, may represent an
optimal synthetic immune index to investigate the functional
status of the anticancer immunity in the single cancer patient,
by representing the synthesis of the actions of the great number
of immunostimulating and immunosuppressive factors involved in
the modulation of the anticancer immunity (Dieckmann
et al, 2001). Within the great number of natural agents
derived from plants and potentially usefull to be employed in
the complementary therapy of cancer, arabinoxylan would seem to
represent one of the potential natural agent, because of its
efficacy in improving the clinical status of cancer patients (Ghoneum
and Jewett, 2000; Ghoneum and Gollapudi, 2005; Markus et al,
2006; Ghoneum et al, 2007). The immunomodulating
properties of this nautral substances extracted from plants have
been confirmed by experimental studies, but unfortunately most
experiments have been limited to the investigations of they
effects on non-specific immune parameters for the anticancer
immunity, such as NK cell cytotoxicity. In contrast, since reg
cells play a fundamental role in suppressing the generation of
the anticancer immunty, each potential antitumor
immunomodulatory natural substances, would have to be
investigated also in relation to their eventual influence on T
reg cell system. On the basis of the recent discoveries in tumor
immunobiology (Dieckmann et al, 2001;
Shevach, 2002), a study was planned to investigate the
possible influence of arabinoxylan on both absolute number of T
reg cells and their ratio with respect to the total CD4+ T cells
in a group of solid tumor patients, affected by locally limited
or metastatic disease.
II. Materials and methods
The study included 24 consecutive patients, 18 of whom had a
metastatic solid tumor, which did not respond to the
conventional anticancer chemotherapies and for whom no other
effective standard treatment was available, while the remaining
6 patients had been surgically treated for a locally limited
neoplasm. Patients were followed at Biological Medical Institute
of Milan and the protocol was approved by the Director of the
Institute. Eligibility criteria were, as follows:histologically
proven locally limited or metastatic solid tumor, no double
tumor, no chronic therapy with corticosteroids because of their
immunosuppressive effects and no concomitant treatment with
other immunomodulating agents,such as interferons,interleukins
and monoclonal antibodies. At the time of the start of
arabinoxylan therapy, patients with untreatable metastatic
cancer were under treatment with the only supportive care,
consisting of anti-inflammatory agents for pain,
anti-dopaminergic drugs for nausea and vomiting and with the
pineal hormone melatonin for the therapy of the neoplastic
cachexia (Banks et al, 1995).
Patients were considered as fully evaluable when they had
received arabinoxylan therapy for at least 2 consecutive months.
Arabinoxylan was given orally at a dose of 1000 mg twice/day for
the first month, followed by a dose of 1000 mg/day for the next
month. Arabinoxylan was supplied by DAIWA Pharmaceutical (Tokyo,
Japan). It was derived from rice bran treated enzymatically with
an extract of the shiitake mushrooms. It is a polysaccharide
containing β-1,4-xylopironase hemicellulose, commercially
available and known as Biobran. For the immune investigation,
venous blood samples were collected in the morning after an
overnight fast before the onset of arabinoxylan therapy and
after 2 consecutive months of treatment. In each blood sample,
we evaluated the absolute number of total lymphocytes, T
lymphocytes (CD3+), T helper (TH) lymphocytes (CD4+), T
cytotoxic lymphocytes (CD8+), NK cells (CD16+ CD56+ and T
regulatory (T reg) lymphocytes (CD4+ CD25+). The different
lymphocyte subsets were measured with a flow cytometric assay by
using specific monoclonal antibodies supplied by
Becton-Dickinson (Milan, Italy). Moreover, because of the
importance not only of their absolute number, but also of their
percentage with respect to the other lymphocyte subsets, namely
to that of CD4+ cells, CD4/CD4CD25 ratio, corresponding to TH/T
reg ratio, was also determined before and after therapy. Normal
values (95% confidence limits) of T reg number and TH/T reg
ratio observed in our laboratory were below 240/mm3 and above
4.0, respectively. Data were reported as mean +/- SE and
statistically analyzed by the Student’s t test, the analysis of
variance and the chi-square test, as appropriate.
III. Results
Evaluable patients were 22/24, while the remaining 2 patients,
both affected by untreatable disseminated liver metastases due
to colorectal cancer, rapidly died for disease progression
before concluding the two planned months of arabinoxylan therapy.
The clinical characteristics of the evaluable patients are
reported in Table 1. Figure 1 illustrates changes in the mean
number of total lymphocytes, T lymphocytes, T cytotoxic
lymphocytes and NK cells occurring after 2 months of
arabinoxylan therapy. No substantial variation was found in the
mean number of lymphocytes, T lymphocytes, T cytotoxic
lymphocytes and NK cells under arabinoxylan treatment. In
contrast, as illustrated in Figure 2, TH and T reg mean numbers
increased and decreased, respectively, after arabinoxylan
therapy, without, however statistically significant differences
with respect to the values seen prior to therapy. On the
contrary, a statistically significant increase in TH/T reg mean
ratio was achieved after arabinoxylan therapy (p<0.025). The
increase in TH/T reg ratio under arabinoxylan therapy was more
pronounced in patients with an abnormally low ratio prior to
therapy with respect to that occurring in those with normal
pre-treatment ratio, however without statistically significant
differences ( 2.3 +/- 0.4 vs 1.7 +/- 0.5). In more detail,
before arabinoxylan therapy, an abnormally low TH/T reg ratio
was present in 12/22 (55%) evaluable patients. Arabinoxylan
treatment induced a normalization of TH/T reg ratio in 5/12
(42%) patients with an abnormally low ratio prior to therapy.
The percentage of arabinoxylan-induced TH/T reg normalization
obtained in lymphocytopenic patients was not significantly
different from that achieved in patients with normal
pre-treatment lymphocyte count ( 3/7(43%) vs 2/5(40%) ). No
toxicity was observed under arabinoxylan treatment, which was
well tolerated in all patients. Asthenia was present in 8/22
(36%) evaluable patients. An evident relief of asthenia, as
assessed by a specific patient report, was obtained under
arabinoxylan therapy in 5/8 (63%) patients.
IV. Discussion
Previous experimental studies had already demonstrated some
immunomodulating properties of arabinoxylan, in particular
consisting of stimulation of NK cytotoxic function (Ghoneum,
1998), whereas NK cell number did not seem to be
influenced by arabinoxylan administration. However, it has to be
remarked that NK cells were belived to be fundamental in the
antitumor immunity until some years ago, before the discovery of
the essential role played by the antitumor cytokines, such as
IL-2 and IL-12 (Whittington and Faulds,
1993) and dendritic cells, because of their function as
antigen-presenting cells (Banks et al,
1995). In fact, it has to be considered that the
cytotoxic activity of NK cells is effective only against
artificial laboratory cancer cell lines, whose biological
malignant properties are different from those presented by fresh
human tumor cells (Whittington and Faulds,
1993). In addition, NK cells have been proven to be also
able to destroy fresh human cancer cells only after the
activation of their cytotoxic function by IL-2 (Atzpodien
and Kirchner, 1990). From this point of view,
arabinoxylan had been already proven to amplify the stimulatory
effect of IL-2 on NK-mediated antitumor cytotoxicity (Ghoneum
and Jewett, 2000). In contrast, no study has been
performed up to now to evaluate the possible influence of
arabinoxylan not only on the mechanisms responsible for the
generation of an effective anticancer immune response, but also
on those involved in the suppression of anticancer immunity. The
results of this preliminary study, carried out to evaluate the
influence of arabinoxylan on T reg cells, which represent the
most important cells involved in the suppression of the
antitumor cytotoxic immune response, demonstrates that
arabinoxylan may counteract T reg cell generation by reducing
their number and percentage with respect to the total amounts of
CD4+ cells and circulating lymphocytes. Since NK cell function
is inhibited by T reg activation (Shevach,
2002), the previously demonstrated arabinoxylan-induced
stimulation of NK cell cytotoxic function might depend at least
in part on its capacity of counteracting T reg generation (Dieckmann
et al, 2001). Moreover, this study would suggest that the
inhibitory action of arabinoxylan on T reg generation is more
pronounced in patients with an abnormally high percentage of T
reg cells prior to therapy, with a following pre-treatment
abnormally low TH/T reg ratio before therapy, whereas its effect
was less evident in patients with a pre-treatment value of TH/T
reg ratio within the normal range. Therefore, the influence of
arabinoxylan on T reg generation would consist of a modulatory
action rather than an inhibitory activity. This finding could
explain a potential favourable immunomodulatory effect of
arabinoxylan also in patients with autoimmune diseases (Ghoneum,
1998), who in contrast to cancer patients would tend to
present abnormally low amounts of T reg cells. In any case, the
importance of the inhibition of T reg generation in the
induction of an effective anticancer immune response has been
recently confirmed by the evidence that the block of T reg
activity by specific monoclonal antibodies may induce objective
tumor regressions in humans (Yang et al,
2007). Obviously, the major problem is the exact
identification of he T reg cell population. Even though T reg
cells may express other immune markers, namely FOX-p2
cytoplasmatic antigen, most clinicians are in agreement to
identify the CD4+CD25+ cells as T reg lymphocytes (12). In any
case, further studies, by evaluating other immune markers, will
be required to better identify T reg cells population, namely
FOX-p3, even though recently some Authors have shown that FOX-p3
expression by T reg cells is associated with a lower suppressive
activity (Dieckmann et al, 2001; Shevach,
2002). Moreover, it has to be remarked that several
patients included in the present study were concomitantly under
palliative therapy with the anti-cachectic pineal hormone
melatonin (Brzezinski, 1997), which
may also play immunomodulating effects (Maestroni,
1993). Therefore, further randomized studies with
arabinoxylan alone versus arabinoxylan plus melatonin will be
required to better define the immunomodulating action of
arabinoxylan. If further clinical and experimental studies will
confirm the inhibitory action of arabinoxylan on T reg cell
system, it could be included in cytokine-based immunotherapies
to enhance their efficacy by counteracting T reg cell
generation.
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