Tumor regressive effects of Viscum album preparations. Exploration of immunomodulatory mechanisms

Abstract

Viscum album is a parasitic plant that grows on apple or pine trees. It is commonly known as European mistletoe. Viscum album (VA) preparations are the aqueous extracts used as a complementary medicine in cancer therapy. Various clinical studies have shown that VA preparations improve the quality of life in different cancer patients. Biologically active components of VA extracts include Mistletoe lectins (ML), viscotoxins, polyphenols and polysaccharides. The treatment with VA preparations or with purified ML has been shown to be associated with tumor regression in several in vivo experimental models of tumoral implantation. Various in vitro studies have shown the cytotoxic effects of VA preparations on various tumor cells. The mechanisms underlying the anti-tumoral activity of VA preparations or ML are complex and involve apoptosis, angiogenesis and immunomodulation. The immunomodulatory mechanisms mediated by the VA preparations can complement the anti-tumoral effects, resulting in effective tumor regression. This review provides an account of the current status of the understanding of the VA-associated immunomodulation in various cell types including lymphoblastoid, monocytic and endothelial cell lines.

Full text

IMMUNOMODULATORY PROPERTIES OF
VISCUM ALBUM 85
International Symposium
NEW DIRECTIONS IN CANCER MANAGEMENT
Academia Nacional de Medicina
Buenos Aires, 6-8 June 2007
MEDICINA (Buenos Aires) 2007; 67 (Supl. II): 85-89
ISSN 0025-7680
TUMOR REGRESSIVE EFFECTS OF
VISCUM ALBUM
PREPARATIONS
EXPLORATION OF IMMUNOMODULATORY MECHANISMS
SRIRAMULU ELLURUa, b, c, d, JEAN-PAUL DUONG VAN HUYENa, b, c, e, BHARATH WOOTLAa, b, c, d,
SANDRINE DELIGNATa, b, c, FABIENNE PROSTa, b, c, VIR-SINGH NEGIf, SRINI V. KAVERIa, b, c
a
Centre de Recherche des Cordeliers, Université Pierre et Marie Curie, Paris;
b
Université Paris Descartes, Paris;
c
Equipe
16-INSERM, Paris;
d
Université de Technologie, Compiègne, France;
e
Department of Pathology, Hôpital Européen
Georges Pompidou, Paris;
f
Department of Medicine, Pondicherry, India
Abstract
Viscum album
is a parasitic plant that grows on apple or pine trees. It is commonly known as Euro-
pean mistletoe.
Viscum album
(VA) preparations are the aqueous extracts used as a complemen-
tary medicine in cancer therapy. Various clinical studies have shown that VA preparations improve the quality of
life in different cancer patients. Biologically active components of VA extracts include Mistletoe lectins (ML),
viscotoxins, polyphenols and polysaccharides. The treatment with VA preparations or with purified ML has been
shown to be associated with tumor regression in several
in vivo
experimental models of tumoral implantation.
Various
in vitro
studies have shown the cytotoxic effects of VA preparations on various tumor cells. The mecha-
nisms underlying the anti-tumoral activity of VA preparations or ML are complex and involve apoptosis, angio-
genesis and immunomodulation. The immunomodulatory mechanisms mediated by the VA preparations can
complement the anti-tumoral effects, resulting in effective tumor regression. This review provides an account of
the current status of the understanding of the VA-associated immunomodulation in various cell types including
lymphoblastoid, monocytic and endothelial cell lines.
Key words:
Viscum album
, cytotoxicity, apoptosis, endothelium, immune system
Resumen
Efecto antitumoral de extractos de
Viscum album.
Búsqueda de efectos inmunomoduladores.
Viscum album
es una planta parasitaria que crece en los pinos y manzanos. Se conoce común-
mente como
European mistletoe
. Las preparaciones de
Viscum album
(VA) son extractos acuosos que se em-
plean como medicinas complementarias en la terapia del cáncer. Varios estudios clínicos han demostrado que
las preparaciones de VA mejoran la calidad de vida de los pacientes oncológicos. Entre los componentes
biológicamente activos presentes en los extractos acuosos de VA se pueden mencionar las lectinas de Mistletoe
(ML), viscotoxinas, polifenoles y polisacáridos. En varios modelos experimentales de implantación tumoral
in
vivo
, el tratamiento con preparaciones de VA o con la lectina ML purificada se asoció con regresión tumoral.
Asimismo, diversos estudios
in vitro
demostraron que las preparaciones de Va tienen efectos citotóxicos sobre
varias células tumorales. Los mecanismos implicados en la actividad antitumoral de las preparaciones de VA
son complejos e involucran apoptosis, angiogénesis e inmunomodulación. Se propone que los mecanismos
immunomodulatorios podrían complementar los efectos antitumorales, resultando en una efectiva regresión del
tumor. Este trabajo revisa el estado actual del conocimiento sobre la inmunomodulación asociada al VA en dife-
rentes tipos celulares incluyendo líneas linfoblastoideas, monocíticas y endoteliales.
Palabras clave:
Viscum album
, citotoxicidad, apoptosis, endotelio, sistema inmune
Viscum Album
(VA) preparations are aqueous extracts
from
Viscum album
or European mistletoe consisting of
different types of lectins1-3. Mistletoe lectin (ML) I, II, and
III belong to the ribosome-inactivating protein (RIP) fam-
ily of type II. RIP of type II are composed of an N-glycosi-
dase (A chain) and a galactoside-recognizing lectin (B-
chain) connected by a disulfide bridge4. The entire prepa-
rations used therapeutically consist in addition to mistle-
toe lectins and viscotoxins, several enzymes, peptides
- - - -
e-mail: srini.kaveri@umrs681.jussieu.fr
(for eg., viscumamide), amino acids, thiols, amines, poly-
saccharides, cyclitoles, lipids, phytosterols, triterpines,
flavonoids, phenylpropanes and minerals. Several stud-
ies have reported the clinical benefits of preparations
consisting of VA extracts in cancer patients5-7. Treatment
with VA extracts or by purified ML has also been shown
to be associated with tumor regression in several
in vivo
experimental models of tumoral implantation8-10.
Experimental anti-tumoral effect of VA extracts may
be supported by
the direct cytotoxic properties of ML to-
wards tumor cell lines1, 11, 12. It is well established that the
cytotoxicity of VA extracts is dependent on the induction

MEDICINA
- Volumen 67 - Nº (Supl. II), 2007
86
of apoptosis. However, the mechanisms underlying the
VA extract-induced apoptosis have not been fully eluci-
dated10, 13-20. More recently, several studies have demon-
strated that VA extracts and purified ML have immunomo-
dulatory properties14, 17, 18, 21-27. VA preparations or purified
ML have been shown to induce activation of transcription
and secretion of pro-inflammatory cytokines such as IL-
1, IL-6 and, TNFα in human PBMC, and endothelial cells17,
22, 24, 26. Moreover, several studies have shown that VA
extracts exert immunomodulatory properties i.e. enhance-
ment of NK cell activity or modulation of TH polarization28,
29. However, the
in vitro
cytotoxic properties of VA ex-
tracts towards cancer cell lines, raise the question con-
cerning the interaction of VA preparations with the sur-
vival of cells implicated in the immune and inflammatory
systems i.e. lymphocytes, monocytes, endothelial cells
(EC). To address this question, we have conducted sev-
eral
in vitro
studies on the interaction of VA extracts with
various established cell lines (monocytic and lympho-
blastoid cell lines, EC lines) or primary cultures (normal
murine splenocytes, HUVEC).
Cytotoxicity of VA extracts towards human
cell lines derived from T and B lymphocytes,
and from monocytes in vitro
Several authors have established that the cytotoxicity of
VA extracts could greatly differ depending on the type of
preparation and to the investigated cell. The induction of
cell necrosis by VA extracts has been reported in human
peripheral blood lymphocytes (PBL), human peripheral
blood monocytes (PBM), murine thymocytes, human
monocytic THP1 cells and mononuclear leukemia cells
MOLT414, 16-18.
We conducted a study to compare the cytotoxic prop-
erties of different VA preparations: VA Qu FrF, Qu Spez,
M Spez and VA P in a large representative panel of
lymphoblastoid cell of T and B origins and in monocytic
cell lines19, 30. The induction of cell toxicity by various VA
extracts was assessed
in vitro
on the human T cell lines
CEM and Jurkat, B lymphoblastoid lines Raji, BC36,
BC28, BC41, WW2-LCL, and in HL-60 and MM-6 mono-
cytic cell lines19, 30. Cell death was measured by the up-
take of propidium iodide followed by flow cytometry analy-
sis. VA Qu FrF, Qu Spez and M Spez induced a dose-
dependent cell death in both T cell lines, and in mono-
cytic cell lines. Cell toxicity was associated with a dose-
dependent inhibition of cell proliferation. As expected, VA
P was devoid of cytotoxicity in all tested cell lines. This
may be in part explained by the low concentrations of
cytotoxic ML in VA P preparations.
The involvement of apoptosis in the cell toxicity in-
duced by VA extracts have been well demonstrated in
various tumoral cell lines13-17, 31. The molecular mecha-
nisms are not fully understood and conflicting results have
been published. Concerning lymphoblastoid cells, using
Fas-resistant HuT78.B1 T cell line, we have demonstrated
that Fas pathway is not involved in VA Qu FrF apoptosis
as previously reported by others32. We have also shown
that VA QuFrF induces a dramatic decrease in the amount
of anti-apoptotic proteins Bcl-2 and Bcl-X proteins in T
lymphocytes19, a finding consistent with the release of
cytochrome from mitochondria induced by ML I13.
At the concentrations studied, nearly all B lympho-
blastoid cell lines were resistant to all the VA preparations
(only BC41 cell line was fully sensitive to the three prepa-
rations) studied. Our findings clearly confirm that cell lines
of B lymphocyte origin are refractory to the cell death in-
duced by VA extracts. Similar results were obtained by
Bantel et al13. The B lymphocyte cell line, BJAB, required
approximately 1000-fold higher amounts of ML-I than that
required by Jurkat cells to attain an optimal level of
apoptosis13. The molecular mechanisms of such resistance
of cell lines of B-cell origin are currently unknown.
Results from preliminary experiments conducted in our
laboratory may provide new insight into the interaction of
VA extracts with the immune system. These preliminary
Fig. 1.– Differential modulation of murine splenocyte
proliferation by VA extracts. Murine splenocytes were
obtained from normal C57BL6 mice (6 mice). Cells were
cultured for 72h in the presence of 50 µg/ml of VA Qu FrF,
Qu Spez, M Spez and P. After 48h, [3H]thymidine (0.5 µCi)
was added to the medium. The cells were then cultivated
for further 24h. The splenocytes were then collected and
the incorporated [3H]thymidine was measured. The results
are represented as a percentage of variation as compared
to untreated controls. After 72h of culture, VA Qu FrF and
Q Spez strongly inhibited murine splenocyte proliferation,
whereas VA P dramatically induced cell proliferation.

IMMUNOMODULATORY PROPERTIES OF
VISCUM ALBUM 87
ex vivo
experiments suggest that VA preparations may
induce cell proliferation in normal C57BL6 murine
splenocytes as measured by cellular incorporation of tri-
tiated thymidine. However, these data clearly show a
balance between inhibitions or induction of proliferation
and induction of apoptosis that varied depending on the
preparation used, the concentration and the incubation
time (Fig. 1). The results emphasize the necessity to de-
sign the adequate schedule to achieve immunostimulation
in both
in vivo
and
in vitro
experiments.
Induction of apoptosis by various
Viscum
album
extracts on both immortalized human
venous endothelial cell lines and HUVEC
In addition to its gate-keeping role between blood and
tissue, the endothelium plays a pivotal role in several
biological processes. EC actively participate in the
regulation of blood flow and coagulation, in initiation and
enhancement of inflammation, and in angiogenesis that
is fundamental to reproduction, development and repair33,
34. The contribution of an EC-mediated effect in the anti-
tumoral properties of VA extracts thus needs to be
investigated. In this respect, we assessed the interactions
between VA extracts and EC.
Our
in vitro
study focused on the ability of VA extracts
to induce EC death. Using primary human venous en-
dothelial cells (HUVEC) and the immortalized human ve-
nous endothelial cell lines IVEC and EA-hy926, we have
shown that VA extracts induce EC death in a dose- and
time-dependent manner20. Fig. 2 depicts the cytotoxic
properties of VA extracts towards EA-hy926 as assessed
by the uptake of PI. Three VA extracts induced a potent
and dose mortality of EA-hy926 as assessed by the up-
take of PI. VA Qu FrF and VA Qu Spez were the most
effective preparations. Iscador M Spez had less cytotoxic
properties. EA-hy926 cells were insensitive to Iscador P
at any tested concentration. Similar results were obtained
with the IVEC cell lines. Other authors have also reported
the sensitivity of various EC lines to VA extracts35.
We then demonstrated that VA-mediated EC death
involves apoptosis, using various methods i.e DNA lad-
der formation, annexin V labeling, and western blot analy-
sis for poly(ADP)-ribose polymerase (PARP) cleavage.
Fig. 3A illustrates the cytological aspect of HUVEC incu-
bated with high concentrations of VA Qu FrF20. Numerous
typical blebs were observed around the cell. As illustrated
in Fig. 3B, HUVEC cultured in the presence of VA extracts
exhibited the typical apoptotic bodies. Annexin V labeling
with flow cytometry analysis and DNA laddering experi-
ments also confirmed the involvement of apoptosis in the
EC cell death induced by VA extracts. The percentage of
EC undergoing apoptosis (An V+/PI-) increased in a time-
and dose-dependent manner for VA extracts. Western blot
analysis also demonstrated that the cleavage of PARP is
involved in VA-induced HUVEC apoptosis.
Can
Viscum album
extracts be used for
anti-angiogenic therapy of cancer?
Angiogenesis is the process by which new blood vessels
are formed by sprouting pre-existing vessels33, 34, 36, 37. Tu-
mor angiogenesis plays an essential role in tumor
progression and metastasis. Clinical applications of research
on angiogenesis have emerged towards the diagnosis,
prognosis or therapy of neoplasia38, 39. The mechanisms
involved in tumor angiogenesis consist of a wide range of
Fig. 2.– Induction of cell death in endothelial cell line EA-
hy926 by VA extracts. Sub-confluent EA-hy926 cells were
cultured for 48h in the presence of increasing concentration
of VA extracts: VA Qu FrF (black bars), Qu Spez (shaded
bars), M Spez (grey bars) and P (open bars), or were left
untreated (Control). Cell death was then quantitated by
measuring the uptake of PI and expressed as a percentage
of dye-positive cells. VA Qu FrF, Qu Spez and M Spez
induced dose-dependent cell toxicity in EA-hy926 cells. EC
were fully resistant to VA P.
Fig. 3.– HUVEC undergoes apoptosis when treated with
higher doses of VA Qu FrF. Sub-confluent HUVEC were
cultivated with VA Qu FrF at 50 µg/ml for 48h. Panel A
illustrates the features of EC under cytologic examination,
with the presence of blebbing around the cells. Panel B
confirmed the involvement of apoptosis with a typical
pattern of apoptotic body.

MEDICINA
- Volumen 67 - Nº (Supl. II), 2007
88
phenomena including enhanced division of endothelial cells
(EC) within the tumor, up-regulation of cell adhesion
molecules and production of angiogenic factors33, 34.
The role of VA extracts in the process of angiogenesis
is thus of interest to understand some of the molecular
mechanisms involved in the anti-tumoral properties of VA
extracts. Fig. 3 illustrates our preliminary results obtained
in capillary tube formation experiments using EA-hy926
cell line. EC were seeded on matrigel and incubated with
various concentrations of VA extracts. A well-organized
capillary tube network was observed in control well (Fig.
4A). In contrast, the capillary tube formation was inter-
mediate with VA P (50 µg/ml) (Fig. 4B), while it was abro-
gated in the presence of VA Qu Spez (50 µg/ml) (Fig.
4C). Thus, these preliminary results suggest that extracts
inhibit
in vivo
angiogenesis in a differential manner.
In
vivo
anti-angiogenesis effect of VA extracts have also
been examined by choriallantoic membrane assays in
C57BL6 mice inoculated with B16-BL6 melanoma cells
and treated with Viscum album L. coloratum agglutinin10.
In addition, VA
coloratum
extracts suppressed tumor
growth
in vivo
and inhibited the number of blood vessels
oriented towards the tumor mass40.
The molecular mechanisms underlying the VA-induced
inhibition of angiogenesis have not been fully elucidated.
At the level of EC, our results may suggest the implica-
tion of VA-associated EC apoptosis, at least in part in its
anti-angiogenic properties. Several studies have well
demonstrated that EC apoptosis is implicated in the physi-
ological inhibition of angiogenesis. Consistently, Yoon et
al. have observed in
in vitro
experiments that VA coloratum
extracts inhibit the proliferation of rat EC.
Conclusions
The molecular mechanisms underlying the anti-tumoral
activity of VA or ML are complex and involve several inter-
related biological phenomena including apoptosis,
angiogenesis and immunomodulation. As in the case of
other members of RIP II family, VA extracts exert cytotoxic
activity towards cell lines derived from both human and
rodent origins although to a lesser extent as compared to
ricin. VA extracts and purified ML also induce activation
of transcription and secretion of pro-inflammatory
cytokines in human PBMC and endothelial cells. To
address the interactions between immunomodulatory and
anti-tumor properties of VA preparations, we have recently
investigated the mechanisms underlying the effects of VA
extracts on tumoral growth of melanoma implanted in
mice. These studies are suggestive of the implication of
certain anti-tumoral cytokines. Together, several lines of
evidence that have been accumulated in the recent years,
in favor of anti-tumoral and immunomodulatory properties
of VA extracts, strongly encourage further intensive
investigation.
Acknowledgements: The authors thank Rainier Dierdorf,
Jean Chazarenc, and Marc Follmer for helpful suggestions.
This work was supported by Weleda AG, Switzerland, and by
Institut National de la Santé et de la Recherche Médicale
(INSERM) and Centre National de la Recherche Scientifique
(CNRS). S.E is a recepient of fellowship from EGIDE, France.
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