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CHAPTER I
INTRODUCTION AND REVIEW OF LITERATURE
INTRODUCTION AND REVIEW OF LITERATURE
Cancer can be described by the definition of the eminent British Oncologist
Sir Hupeo W~l l~s as an abnormal mass of tissue, the growth of which exceeds and
~s unco-ord~nated with that of the normal tissues and persists in the same excessive
manner after the cessation of the stimuli which evoked the change (1). Neariy a
cerllury of surgical audacity and ingenuinty, coupled in last 40 - 50 years with the
advances in anticepsis, antibiotics, blood replacement and intensive care have
greatly increased the resectability of primary cancers. However, too frequently, the
catlcers recur locally, regionally or systemically and the patient eventually succumbs
(21.
n the United States, cancer is second only to heart disease as a cause of
dea!li and accounts lor 22 per ceht of all deaths. Alnxst one million newly diagnosed
cases of cancer and nearly 5.00,OOiJ deaths due to cancer were predicted for the
United States during 1988 (3). In India it has been estimated that there is about
! 5 rniliior~ cases oi cancer in the ccunlry a1 any giver1 tirne with about 0.5 million
new cancer cases being added every year. Morc lhan 60 per cent of these affected
patients are in the prime of their life between the ages of 35 - 65 years. When
this s~tuation is projected to the year 2000 AD, the population with cancer is likely
to beco~me far Inore acute than it is today, with the increasing life expectancy and
charlging lile style concomitlant with development, the number of cancer cases wiil
be aIr.cs! :mice tha currerit ciilniber. All the centres in india as well as in abroad.
reported an increase in year-wise incidence of cancer, which may be attributed to
the improving diagnostic and reporting facilities (4).
The etiopathcgenesis of neoplastic disease is charaderised by its muniple natu-
re. Biological (5), chetmicai (6) and physical (7) agents have been identified as initia-
ting or p~omoting neoplastic mechanisms. However, they all appear to have a corn
mon molecular basis causing genetic instability followed by somatic derangements
to preneopiastic and neoplastic cells. In additon to these somatic mutations, which
are the most frequent abnormalities identilied in human cancer, the germ-line muta-
tions associated with specific familial cancer syndromes have also been recently
character~zed. Epidemiologic and molecular genetic studies have unveiled the
underlying mutations of speclic genes, predisposing palients to distinct cancers such
as intraocular tumur and retinoblastoma (8, 9). it is, therefore, conceivable to view
cancer as iundamentally a genetic disease entailing germ line and somatic
mutations.
Target genes implicated in cellular transformation and tumour progression have
been divided into two categories viz. proto-oncogenes and tumur suppressor genes.
Activatior~ of proto-oncogenes by either point mutation, amplification, translation or
even insertion of noneukatyotic sequences yield oncogenes that have as main charac-
teristic a "gain' of function. They have also been called 'dominant' and 'positive'
phenomena. Inactivation of tumour suppressor genes occur mainly through an allelic
deletion followed by a point mutation c! the con:ral;teral alle!e. These events also
have known as 'recessive' and 'negative'. Complt.xation of some tumour suppressor
gene products by other miecuizs ma{ render them inactive, having a similar final
effect as inactivation by means of a genetic abnormality. Alterations in proto-
oncogenes and tumour suppressor genes seem equally prevalent m n g human
cancers (lo. 11).
Scientific understanding of neoplastic disease has generated an impressive know-
ledge base related to the fundamental biological mechanisms undellying cell growth
and reyvlation and the management of this devastating disease. The management
considerations that are important in formulating a data-base for individual cancer
patient include detection, establishmnt of histopathobgic diagnosis and staging of
the disease. After defining the status of the tumour and the therapeutic goal, a
treatrnent plan is formulated. The important therapeutic modalities currently available
include chemotherapy and irnmunotherapy in combination wlh other modalities of
treatrnent like surgery or radiation. But all modalities of cancer treatment have certain
limitations. Invariably in all treatrnent mdalities, normal cells are also affected to
a greater or lesser extent. Even Vlough chemotherapy is a relatively new everil
in the treatment of cancer, the impact of this modality is increasing exponentially.
During the last 30 years, chemicals of every conceivable naturc have been listed
as potential anticancer agents. These include poorly charactcrued biological extracts.
membrane active agents and a plethora of drugs which were originally developed
lor another purpose. Antiproliferative agents which had the theoretical advantage,
tha: their side effects are directed primarily towards the proiiferating tissues succee-
ded in this effort. Unfortunately, chemotherapy suflers from an insufficient selectivity
lo1 !tie target turnours anc toxicity to normal tissues.
Many approaches are being pursued to develop more effective anticancer treat-
ment and these include the development of new d ~ g s and new modalities of treat-
ment based on increased knowledge of the biochemical and biological basis of selecti-
vity of antitumour action as well as the development of new types of treatment
that may ~mdi ly the interaction between host and turnour. Cancer treatment that
produces antitumour effect thmugh the adbn of natural hostdefense mechanisms
or by tha 3Jministration 01 natural mammalian substances is biologic therapy. Among
the different modalities of cancer therapy, biologic therapy has emerged in the last
several years as an impoltant fourth mdality for the treatment of cancer.
With the development of in vitro and in vivo screening procedures (12. 13)
several naturally occuring compounds and thousands of plant extracts are tested
each year for their potential use as anticancer agents. Taxol, a Diterpenoid isolated
from Taxus breviiolia (14) has been reported to possess antineoplastic activay (15,
16). The vinca alkaloids Vincristine and Vinblastine (17, 18) are used in combination
with bleomycin and cisplatin for the therapy of metastatic testicular tunlours (19).
Two glycosidic derivatives of podophyllotoxin, VM 26 and VP 16, are found to have
significant chemical activity against Hodgkin's disease, small cell carcinoma of the
lung and .c.sticular tUmOUrS (20). Curcumin present in turmeric is a strong antioxidant
and anli~nflammatory reagent (20, 21). It is also known to scavange reactive oxygen
species (21) and inhibit chemical carcinogenesis in experimerital animals (22, 23,
24). Anti-carcinogenic natural product, isolated from edible umbelliferous plants has
been found to be a promising chemprevcnlivo agent (25). Similarly Brassinin, a
natural product found in Chinese cabbage is a ruvel chcrnopreventive agent inhibi-
ling the development of mammary lesions in a dose dependant manner (26).
While so much is known about the protective and therapeutic use of variouS
plant agents in mitigating the severity of cancer, there are repons that an aqueous
extract from European mistletoe (Viscvm album L) has been used for the treatment
of human neoplasm since in the early twenties wilh much advantage (27). The
extract of mistletoe has been tound to be cytotoxic to various cell lines and also
inhibited experimental animal tumours (28, 29). The mechanism of action includes,
stimulation of cell mediated immune responses as well as increased rnaturatwn and
prolilerat~on of mnocytes (30, 31, 32). Inspired by the bcneiicial and encouraging
results obtained on the administration of the extract of European semiparasilic plant
Vissum Jmn (family Loranthaceae) in ameliorating neoplastic diseases, an experi-
mental programme has been conceived by utiliuing semiparasitic plants available
in Kerala (India) to investigate in depth, the following technical aspects.
1. Cytotoxic and antitumur activity of selected medicinal plants, especially from the
family Loranthaceae.
2. Tumour reducing activity of these plants by using animal tumurs
3. Isolation of the active ingredients from these plants by chmmatographii
methods.
4. Biochemical and immunological mechanism of action.
5. Chemoprotectire and radioprotective action.
In cruer to fulfil these objectives, the semiparasitic plants of the family Loran-
thaceae grown on different host trees in and around 'Thrissur (Kerala) was collected
and they were authenticated by a Botanist. Using these plants, extracts were prepa-
red and the cytotoxic and antiurnour potential was evaluated using dmerent cell
lines in different modalities, Inspired by the positive results of the cylotoxlc and
antiturnour activity of borne of these plant extracts, investigatbns were further exten-
ded to the nature of the active component of this extract and its b i i m i c a l and
immunological mechanism of ar%on. An insight was also made into the radiopmlec-
five and chemprotective action ot the extract. Incidentally, a lectin was also isolated
from certain fractions of this extract and the cylotoxic and antitumur potential of
this lectin was also evaluated.
1.2 REV1 : W OF LITERATURE
Over the centuries concepts of cancer arose that were proposed as means
of explaining the disease to satisfy the physicians of the day. These explanations
were restricted to the contemporary philosophic, theologic and scientific knowledge
and as a result, a serles of conceptual theories of cancer such as theories based
on Egyptian Papyri (33, 34)" Greek concept (39, Humoral theory of cancer (36),
Lymph theory of cancer (37), Blastema theory (36,37), Embryonal theory (37), Mecha-
nical isolation theory and the Traumatic origin of cancer (36, 37) and Parasitic theory
of cancer (34, 36) have been proposed, but if studied carefully m y provide insight
into soine of our scientific controversies. Attainment of the best treatment of cancer
will continue to be of paramount importance for the forseeable futura. One of the
cttrien: ioymas, almost a cliche, is the importance of integration of dierent treatment
modalities viz., surgery, radiotherapy and chernctherapy. Yet this is still often much
more precept than practice. A review confined to these aspects is an attempt towards
achieving this important goal.
1.2.1 Etiology of cancer
A large number of agents induce neoplastic transformation of cells in vitm and
cancer in experimental animals. They can be divided into the following categ~ries.
1. Chemical carcinogens 2. Radiant energy 3. Oncogenic viruses and 4. Foreign
body carcinogens. Certain chemical carcinogens and radiant energy are the doarmen-
led causes of cancer in h I J ~ n s and the evidence linkirg viruses to certain types
of clinical neoplasia grows ever stronger. Epidemologic studies hint strongly that
80 or even 90 per cent of human cancers are the result of life style and other
environmental influences (38). H is an important factor to be considered that several
agents may act in concert, or synergbe the enects of others. Neoplastic translorma-
tion is a progressive pmess involving multiple steps and the chemical carcinogens
may in it at^ the process that is completed by oncogenic viruses (39). It is possible
therefore, that at least some cancers in humans are the consequences of unfortunate
confluence of several of the carcinogenic influences.
1.2.2 Attributes of transformed cells
When normal cells are exposed in vitro 13 carcinogenic influences (chemical
carcinogens, oncogenic viruses, radian1 energy) they acquire many altered characte-
ristics involving growth, behaviour and morphology and are said to have undergone
transformalion. The molecular event or events that bricg about the conversion of
normal cells to cancer cells is of course a critical issue that is still under invesliia-
lions. However, the genetic mechanisms underlying tumorigenesis can be summari-
sed in the lolbwing principles (40).
I . Human tumours represent the clonal expansion ol a single transformed cell. The
transformation results from the acquisition of one or more mutations in at least one
tumur suppressor gene.
2. The tumours progress in association with, and presumably because of rmtat'ons
in other suppressor genes and oncogenes.
3. These mutations occur in a preferred order as turnour progresses from benign to
malignant stage.
4. Mutations in the same nucleotide genes in the same positions can occur through
either inherited or somatic pathways. Inherited cases are responsible for familial
cancer predisposition. Such families have a high propensity for cancer formation
because they transmit in their germline, a mutation in tumocr suppressor gene.
5. Mutatiorr in turnour suppressor gene result in the bss of biological and biochemical
activities associated with the wild type gene product. Oncogenes normally promote
cell growth under appropriate circumstances.
6. The pattern of genetic alterations in tumour can be used to help predict their biologic
properties and propensity to metastasize.
7. Mutant genes can be used as markers of inheritod risk (germ line mutations) or
as markers for the presence ol occult tumours (somatic mutations).
Oncogene relatlon to cancer
Oncogenes are genes that are capable of indudng or maintaining ce!l transforma-
tion and they were discovered through the study of transforming retroviruses. The
proto-oncogenes are normal cellular genes which are the progenitors Of oncogene.
The oncogenes are anered or over-expressed versbns of their normal cell~lar proto-
oncogerni: counter parts. Oncogene encode proteins called oncoproteins which are
very s~milar lo :he normal product of protooncogene except that they have bsl
important regulatory constraints on their activity and they don't need external act'wa-
tlon signals.
1.2.3 Biological aspects of cancer cells :
All tumours, benign and malignant, have two basic components (a) prolierating
neoplastic cells that constitute their parenchyma and (b) supportive stroma made
up of connective tissue, blood vessels and possibly lymphatics. AillNUgh, paren-
chymal cells represent the proiierating cutting edge of neoplasms and so determine
their nature, tne growlh and evolution of neoplasms are critically dependant on their
stroma. An adequate stromal blood supply is a pre-requisite and the slromal connec-
tive tissll. pr~vides the framework for the parenchyma. In some tumurs, the stmmal
suppcrt is scant and so the neoplasm is soit and fleshy. Sometimes the parenchymal
cell stirrwlates the formation of an abordant collagenous stroma referred to as
desnmplasia showing stony hard mass or scirrhous (41).
Characteristics of benign and maligent neoplasms :
Dlfferentlatlon and anaplasla
Dilferentiation and anaplasia apply to the parenchymal cells of neoplasms. Diffe-
rentiation refers to the extent tc which parenchymal cells resemble comparable IlGr-
ma1 cells, both morphologically and lunclionally. Well differentiated tumours are thus
composed 01 cells resembling the mature normal cells of the tissue of origin of
the neoplasm. Poorly differentiated or undifferentiated cancers have primitive-
appeari.:j unspecialiued cells. Maliinant neoplasms range from well diiferentiated
to ~rndilferentiated. Anaplasia is a characteristic of cancerous cells and so constitules
one of the ieatures that makes tumour as malignant. It implies some lack of diierentia-
tion of tumour cells. Lack of dinerentiation or atlaplasla is marked by a display
of pleomrphism - variation in size and shape characteristically the nuclei contain
an abundance of DNA and are h~perchrornalii during staining. The nuclear
cytoplasmic ratio may approach 1:1 instead of the normal 1:4 or 1:6. The nuclear
shape is extremely variable and the chromatin is coarsely clumped and distributed
along the nuclear membrane. Another important feature of malignant neoplasm is
atypical and bizarre mitotic figures sometimes producing tripolar, quadripolar or muili-
polar spindles.
F - -~~.-.ton microscopic, histochemical and immunocytochemical studies have eden-
dea to the characterization of neoplastic cells (42)
.7ate af Growth :
G~owlh rate of t u l rw r j correiate with their level of differentiation and most
malignant turnours grow more rapidly than benign lesions. Many influences involving
the neoplasm and host modify the growth rate of cancers. Blood supply, nutrition,
delensive immune response of the host, and in some tumurs endocrine support
are the most important influences. Fohnan and collaborators have demonstrated
that the development of an adequate vascular supply is critical to growth and devebp
rnent of a cancer as well as its metastases (43). Later it has been found that
neoplastic cells elaborate a soluble tumur angiogenesis factor (TAF) and promotes
vascularization of the stroma and permits the progressive growth of solid tumoun
(44). Cancer cells in their growth are also dependant on an adequate supply of
nutrients and when deprived, continue to replicate lor a few days and then rapidly
die (45).
Encapsulation - lnvaslon :
All benign tumours grow as localized expansible masses enclosed within . fi-
brous molecule. They remain localized to their site of origin and cannot disseminate
throughout the body. Cancers are never encapsulated and are characterized by
infiltrative, erosive growth that extends crablike feet into adjacent tissues. Histological
examinatiol: disclose tiny pseudopods indicative of penetratir~g spread. Most cancers
are invasive and recognize no normal anatomic boundaries and often permeate lym-
phatics, blood vessels and perineural spaces (41).
hietastasis :
Metastases are tumour iniplants disconlinuous with primary tumur. It is a
cascade of linked sequential steps traversed by tumour cells. In order to be succesful
a mela:.atic tumour leave the primary tumour, invade local host tissue, enter the
circulation, survive in the circulation, arrest at the distant vascular bed, extravasate
into the organ interstiiium and parenchyma, and mukiply to inliate a metastatic cobny
(46). Cells obtained from individual tumours have been shown to diier w lh respect
to the~r antigenic properties, karyotypes, growth rate, hormone receptors, ability to
invade and metastatic capabilities (47). This heterogenlty is linked to tumour progres-
sion. Progression implies that over the cwrse ot I s evolution a cancer or particular
subpopulation or clones acquire an increased growth rate or the capacity to invade
or to metastasize.
1.2.4 Immunology of tumour cells :
The concept that immunosurveillance protects tumour cell transformation that
occurs constantly and at hgh frequency was first suggested by Eht i ih in 1909
(48). With the demonstration that chemically induced tumours were strongly antigenic
in the autologous host (49), it became obvious that certain tumours carried tumour
specific or at least turnour associated antiiens.
Tumour antigens :
The two best studied tulnour antigens are alphafetoprotein (AFP) and carcinoem
bryonic antigen (CEA). Alphafetoprotein is a serum protein associated with normal
fetal and neonatal development and with the growth of hepatocellular tumours (50).
Carcinoembryonic antigen is a fatal colon cell-surface glycoprotein that is produced
by tumours of ectodermal origin intestinal, pulmonary, pancreatic, gastric and mamma-
ry ader::arcinoma (51). Elevated serum CEA levels ate also associated with inflam-
matory aiseases of bowel, lung and pancreas. Turnour-associated an~gens and thek
corresponding antibodies play a role in the way In which tumurs evade lhe hosts
immune system (52).
Evaslon of speclflc tumour lmmunlty :
Masking of cell surface antlgens:
One of the simplest concepts of tumour escape from specifically immune lympho-
cytes was based on the evidence that certain mlecules such as sialomucin which
lrequently bound lo the surface of tumour cells are able to mask turnour antigens
from complete exposure in vivo and also to prevent adhesion of sensitiued lympho-
cytes (53). The cell surface sialic acid content varies during the cell cycle (54) with
the hig: t st levels in mitotic and transformed blast cells (55).
Shedding of turnour antigens
The failure to elicit an immune response to a tumour in an imm~nol~gl~al ly
competent host may be resuiling the induction of tolorance to tunmur specli
antigens. Acquisition of tolerance renden normally antigen reactive T and I3 cells
insensirive to the appropriate activation signals by either altering the cell's reactivity
to antigen, saturatiw anligen receptors on the cell surface with antigen of b w irnnuno-
genicity and thus preventing stirnulaton of the cell by highly immunogenic antigen
or the elimination of specifically reactive clones through direct antigen contact (56).
The presence of circulaling soluble tumur antigens has been demonstrated in the
sera 01 turnour bearing animals (57) as well as that of humans (56). The biological
role oi shed tumour antigens proposed by Curie and Alexander was to assist
metastic spread ot the tumour by saturation with the effector limb of T-cell mediated
immunity in tumour micro environment (59).
Blocking factors In turnour envlronrnent :
The shed antigens play an important role in enhancing tumur growth by interac-
llon wit!^ the host-produced specific immunoglobulin to form antigen - antibody corn-
plexes The factors produced in the serum of tumour-bearing host can positively
block : 1 ioloxic activity by autologous immunocompetant cells (60).
Another possible mechanism of antibody mediated escape may involve the con%
plete or partial loss of turnour antigens or the suppression of their expression in
viva as a resun of antigenic modulation (61). 'The aritigenic modulation involves
antigen shedding, endocytosis and redistribution within the cell membrane without
a complete loss of determinants from the cell surface.
1.2.5. Interaction between neoplastic cells and the host's lmmunoreyuliitory appara-
tus :
T t ~ e inimunoregulatory functions have been found to be quite complex. The
piod~it; of ail enector arm of the immunologic network such as cytotoxic antibody
or cGolytic T Celts can regulate the entire immune system by feed back inhibition
augmentation of the induction phase of the specific immune response. The ultimate
expression of specific immunity whether to a simple hapten or a transformed and
proiiieraling tunlour cell popc~latior, is thas the c!ldpoin: of the interplay between
pos~!ive and negative regulatory interacticris. Ant iWy itsen may exert feed back
control lor its own production by binding specific circulating antigen and eliminating
immunologic stimulus (62). Antigel1 - anlibody complexes may directly block the
receptor sires on aflerent immunocompetent cells (63) or may induce toleran~e of
the specific response alter binding to aflerent or efferent receptors (64). Feedbadc
signals may also be delivered by effector T cells and may stimulate either suppres-
sion or activation of the specilic cellular response (65). Phagocytic macrophages
can exen a dual role both as effector cells and regulatory cells by delivering either
act~vatii!g ar suppressing signals to the irnmunoreguiatcry network (66). Moreover.
each of these feed back signals itself is rigorously controlled in that it may act
in a non-specific fashion (67), may regulate a paiticulaf immunoglobulin isotope (68),
niay exillbit genetic restriiions (65), or may reg~late only the idiotype-specilic respon-
se while having no effect on other antigenic responses (70). Hence the mechanisms
by which 1umourS avoid immune dest~ci ion are very complicated and is dosely
linked to the host's immunoregulatory apparatus with potential interactions involving
tumours. specific antibody recognition and eflector lymphocytes, macrophages and
inflammatory factors. The successful application of immur~olherapy to neoplastic
d~sease is criiically dependant on these immunoregulatory intaractions between
t i~ iwur and host.
Many studies have demonstrated lhai the turrour bearing hcst is in~rniinosuppres-
sed by i l ie c;rowing tumou: and in?n:unotherapy refers to the 1:eatment of cancer
!Flat prodbces anttucnour effects pii~1a:i:y through the action t i natural host defense
mechanism or by the administration of natural mammalian substances. Most efforts
to ut~l~ze ~mmunotherapy for the treatment of cancer have involved attempts to stirmla-
te immune defense mechanism by various biologic response modifiers (BRMS). Stra-
tegies for the immunotherapy of cancer can be divided into active and passive
approaches. Active imrnunotherapy refer to the immunization of the turnour bearing
host with materials designed to elicit an immune reaction capable of eliminating
or retarding turnour growth. Active immunotherapy is further subdivided into nonspeci-
fic or specific immunuation. Nonspeclic active immunotherapy utilizes the stimulation
of the immune system with adjuvants such as BCG, C. pawum, levamisole and
a variety of other substances. Treatment with the interferons or with IL-2 is also
a lorm of nonspecific active immunotherapy.
Active nonspeclflc lmmunotherapy
The agent which can alter host-turwur relationships in favour of the host can
be conslclered as a biologic response modifier. Most biologic response modifiers
stlnluialu the Immune system and the most commonly utilized agents in biologic
therapy include microbial augmenting agents, macrophage activators and interferon
inducers, cytokines and irnmur~omodulators.
Mlcroblal augmenting agents
The largest body of data of BRM is relating to Bacillus Calmette Guerin (BCG).
More than a decade ago it was discovered that BCG had a significant antiturnour
acrivdy against a wide variety of neoplasm (71, 72). The efficacy of BCG for treating
neoplas~a IS related to the ability of the microbe to non-spocificaliy stimulate the
immune system. Bacillus Calmette - Guerin enhances resistance lo intracellular para-
meters, antibody production and cellular Immunity as reviewed by Laucius (73) and
Mitchell and Murahata (74). Among the earliest demonstrations of the antitumour
effects o! BCG were the animal studies of Zbar and associates (75). work in^ with
an inbrea strain of guinea pigs and a transplantable hepatocarcinoma, these investiia-
tors demonstrated that intralesional BCG can induce regression of established dermal
tumur transplants and microscopic regional nodal metastasis as well as stimulate
lumur specific cell mediated immunity. BCG modulates a wide variety of immunofunc-
tions and the antitumour etfect is mainly due to rnacrophage activation.
The ~mrnunologic effects of Corynebacterium parvurn are diverse (antibody pro-
duction, delayed hyper sensnivlty, antiiumur Immunity), lnvolvlng modification of
several cell types (macrophages, T-lymphocytes, B-lymphocytes) and may be posi-
tive (stimulatory) or negative (inhibitory). Berd (76) has extensively reviewed the
effects of C.pawum on various aspects of immunity.
Synthetf: rnacrophage actlvarors and Interferon Inducers.
One of the maior mechanisms of action of the microbial adjuvards is activation
a! effeaor cells such as macrophages, natural killer cells and cells that mediate
ADCC. These activiiies are induced at least in part by the generation of interferon.
h large number of agents have been shov~n to be capable o: inducing IFN production
in animals (77) including - sy~thetic polyribonucleotides, fungi, fungal extracts and
piodu~ts, whole bacteria and bacterial products, chlamydia, rickettsiae,
rnycoplasmas, protozoa, miloyeris and a variety of synthetic inducers including anio-
riic polymers and low molecular weight inducers such as tibrorle, and cationic dyes
including quinacrine and various substituted pyrimidines (78). A class of p0lymerS
that mimic viruses and are potent interferon inducers include the synthetic double
stranded RNA group based on Poly I - Poly C (Poly IC). They include Poly IC,
Poly !C stabilized with poly L lysine (Poly ICLC) and Poly IC with mismatched bases.
These in general induce interferon, activate macrophayes and NKcells in a variety
oi species and have viral, protective and antitumour activities (79). Another inte-
resting group of polymers consists of MVE series ol maleic anhydride divinyl ether
plylmers of defined molecular weight and are weak interferon inducers, but have
poient viral protective and arititumur activity (80). A number of low molecular weight
substances have also been shown to be inducers of interferon. These include hetero-
cyclic bases such as acyridine orange, methylene blue and a number of quinolines
and arrLl-iraquinones, the fluorenones such as tilorone; the lipoidal amines, the pyrimidi-
ne der~vatives and certain thiols (81). Another group of agents related to the macro-
phage activators are the lysolecithin analogues (82). The investigation of Modolelt
el 31 on non-hemolytic lysolecjthin analogues showed that they are potent macmpha-
ge activators to boost cellular immunity (83).
Cytoklnes
Cvlokines may be defined as effector or regulatory molecules normally released
fronl activated cells that affect the bahaviour of other cells i r ~ the immediate envimn-
rnent cr at a distance. They tray afiecl cells oi the saxe ot of a different tissue.
'They are usually glycoproteins and interact witn cell surface receptors resuiting in
changes in cyclic nucleotide metabo!isn: and electrolytic flux. Cytokines with anttu-
mour activity include the interferons. Extensive work done by Papermaster et a1
(84) on lymphotoxin which is included in the crude or partially purified lymphokine
preparations derived from the supematent of 1788 cell line by continuous proliferating
Bcell lines demonstrated to have both MAF (macrophage activating factor) and MIF
(migral~on inhibitory factor) and chemotactic and lymphotoxin activity and not to con-
tain interferon. Preliminary studies in mice have shown that this peparation can
produce progression of L1210 leukemia and prolongation of life span in tumur
bearing animals. Another important antitumour cytokine is tumour necrosis factor
(TNF), a glycoprotein with a molecular weight below 70.000 that can be obtained
from the serum of endotoxin treated mice previously immunized with BCG (85).
It is produced by monocytes and may be referred to as monokines. Tumour necrosis
factor can cause the in vivo necrosis o! animai tumours and in vitro is toxic to
animal and human tumur cells but not to normal cells. Lymphocyte activating factor
(LAF) is a monokine produced by activated monocyles (86) known at present as
interleukln 1. It's activity is to augment the short t e n T-cell proliferation in response
to PHA and ConA and to augment the antigen dependant cell mediated and humoral
respo1:ses. T-cell growth factor, now referred to as interleukin - 2 (87) is a lyrnphoki-
ne. oer>:;d from PHA and ConA stimulated lymphocyte wlturc supernatents and
mors. lecently from certain lymphoid cell lines. it is absorbed to specific receptors
on T-cells and can stimulate continuous T-cell proliferation after mitogen or antigen
Stimulation and stimulates the production of specific cytotoxic T lymphocytes to cells
conta~ning the inducing antigen on their suliace. In the continuous presence of T-cell
growth factor, clones ol specific T lymphocytes can be produced and they could
oe ut~i~zed for antltumour therapy. Certain lyrnptiokines play a role in stimulation
oi bone marrow proliferation. These include colony stimulating factor (CSF) and
other colony stimulating or differentiating factors (88) that could be utilized to protect
the bone marrow from the damaging effects of chemotherapy and radiotherapy.
Adoptive lmmunotherapy
Adoptive irnmunotherapy implies the transfer of imnxlnocompetence or specific
turnour ~mmunity from one individual to another. Transfer can be xenogenic, allogenic
or syngerieic and can involve intact cells or subcellular fractions. Bone marrow tfan-
splantdtion as a treatment for malignancy can be considered as an approach to
adop!ive dnmunotherapy. The !ow molecular weight dialuatle extract of peripheral
blood ly~nphocytes consisting of a complex of peptides and nucleic acid, generally
known as Yransfer factor' can transfer specific microbial immunity from an immune
io a nonimmune subject (89). The transfer iactor can have protective andlor therapeu-
tic activity in a variety of human viral and fungal infections. Another approach to
adoptwe immunotherapy is by immune RNA. lmmune RNA can transfer various
types 01 immunity, including tumour immunity from the lymphoid coils ol immunued
allogeneic or xenogeneic subjects to non-immune subjects (90). Tumour immunity
transferred with immune RNA in animal models can be tumour preventive and
can have true imrrltinotherapeutic activity causing either tumour progression or preven-
tisn of lumour recunence afler surgical extirpation of disease (91).
Active S!~cclfic immunotherapy
Active specific immunotherapy is defined as immunization of a subject with
turroui cells ar tcrnour antigen preparations, the cbjective of which is to boost tumour
specific ~mmunity and to allow the generated antibody or effector lymphocytes to
kill tumour cells. Scientifically it has been difficult to establish that there are induced
turnour specific antigens and tumur-specific immune responses associated with
human malignancies. However, the recent work of Pfreundschuh et a1 (92) with
natural antibody and of several investigators with mnoclorial antibody (93) have
identified these antigens. Theoretically, M the appropriate tumour specific immunity
could be generated, eflective control of tumour growth couW be achieved which
had been demonstrated by animal studies (94).
Passlve lmmunotherapy
Passive imrnunotherapy or serotherapy may be defined as the administration
of antltumour antibody to cancer patients, generated by various methods with the
intention of causing tumour regression or preventing tumour recurrence (95). The
development of cloned B-cell populations producing highly specific high titer monoclo-
nal anl~body and the potential for developing human myeloma-lymphocyte hybrido-
mas makes far reaching results for passive immunotherapy.
The irnmunomodulators have a biphasic effect on the immune response either
by stimulating or inhibiting the immune response and other host defense rnecha-
nisms Tttey stimulate the individuals whose host defense parametars are deficient
or low 2nd inhibit host defense parameters in individuals in whom they are normal
or already activated. Biologic therapy agents described earlier such as augmenting
agents, microorganisms and their piod~C!S, chemical compounds and physiologic
mediators can exhibit this basic function. Thymic hormones have also been utilisecl
to produce immunorestorative enecl In animals rendered immunodeficiency by the
Induction of turnour. Thymosln traction - 5 has been demonstrated to have therapeu-
tic efficacy in T-cell deficiency diseases such as the Digeorge syndrome, the Nezalofl
syndronie and the Wiscon Aldrkh Syndrome (21). Both TPI (thymostimrlin) and
THF (Thymus hormone factor) have been shown to have therapeutic acliity in
patients with underlying malignancy who have T-cell deticiency and disseminated
viral inlection (96).
lmmunomodulators of plant origin
Several plant products which can stimulate the immune system specitically and
non-specitically have been isolated and characterized (Table 1 . I ) . They include seve-
ral plant extracts with unidentified structures. These plant products are less toxic
when compared to synthetic drugs and can be administered continuously without
much s c'e effects. Many of these plant products have anticancer activities (97 -
101 129) and are used in cancer therapy.
~ab ' le 1.1
Plant lmmunomodulators with anticancer activity
Name of the plant lmmunomodulatory activity
1. Panax ginseng
2. Trichosanthes rhizome
3. Traxacum platycarpurn
4. Pine cone
5 . Paris forrnosana hyata
6. Viscurn album
Phagocytosis, antibodyproduction. lncrease
in serum complement content, IgG level, B
celVT cell ratio.
Increase in leukocyte counts, peritoneal exu-
date cells, antibody forming activity.
Increase in peritoneal exudale cells.
Peripheral plaque forming cells, induction of
cytokine production (TNF), macrophages,
PMNS and splenocytes were stimulated.
Enhancement of PHA stimulated peripheral
whole blood rproliferation, NK-cell activity,
IFN induction.
Peripheral plaque lorming cells, macrophage
activalion, NK and ADCC aclivity.
Role of lmmunomodulators In cancer therapy
With an established tumour, the imnwne response of the host will be too meagre
lo ablate all the malignant cells. In the conventional therapeutic modalities of cancer
l~ke surgery, radiotherapy and chernotherapy, the immunological surveillance (102)
ol the cancer patient is considerably suppressed (20). The maintenance of the immu-
nological status of the patient is of major importance for the control and cure of
cancer and also to protect the body from other secondary infectious diseases resul-
ting lrom immunosuppression.
Va:ious immunomodulators are now used along with chemotherapzutic agents
(103). The pharmacology and pharmacokinetics of drugs are affected by these materi-
als. BCG or C. parvum can inhibl drug metakalizing activity in the liver (104). The
metabolism of drugs like cyclophosphamide, which depend on liver microsomal activa-
ilon can be modified by irrununotherapy (IO5j.
1.2.7 Lec t i ns
Lectin is a camhydrate binding protein or glycoprotein of nonirnmune origin
which a9glutinate cells or preciplate glycownjdgates or both (106). Lectins contain
atleast two carbohydrate binding sites. The specificity of a lectin is generally presen-
led irl +;ms of the monosaccharide(s) or sirnple o!igosaccharide that inhibits the
lectin associated reaction. Lectins may be deiived from plant, rnicrobial or animal
sources anti :iiay be soluble or membrane hunci. Lediix may also bind to noncah-
hydrare ligantis (107) and have been stac.,vn to react wi!h nuclei (108). Most cornnun
lectins are derived from plant seeds, bark, tubers, pulp, bulbs and leaves and they
have been purified from crude agil6ous, butfer or saline extracts by the methods
01 purification 01 protein. Lectins are also found in othe: biological sources including
bacteria, invertebrates and mammalian tissues (109). Lectin-call binding can elkit
a wide variety 01 biological phenomena. Lectins induce mitosis in resting lymphocytes
and some agglutinate neoplastic transformed cells, but not their healthy non-
malignant counterparls, whereas others agg!utinate heaithy non-malignant cells (107,
110). Lectins have been used to fractionate animal cells, including B and T lympho-
cytes (1 1 I ) and to demonstrate changes in cell surface architecture f ~ l l ~ ~ i n g V~NS
infect lo;^ or parasite infection (107). Some lectins are poterlt toxins and have been
applied as therapeutic agents. The lectins, ricin and abrin, have been coupled to
specific monoclonal antibodies and applied as imrnundoxins in cancer therapy. Other
cytoloxic lectins are modeccin, viscumin alxl voikensin which are isolated from Ade-
nra digrtata, Visarm album leaves and Adeniz mlkens9 roots (112). These lectins
are closely related structurally and functionally. Ricin, the widely studied lectin, is
a heterodimer comprising of two distinct N-glycosylated polypeptides joined by a
disulfide bond. One polypeptide (A chain) is an enzyme which irreversibly inactivates
60s ribosomal sub units. When mammalian ribosomes are exposed to ricin A chain,
the pcoidn-synthesis stops (113). The second ricin polypeptide (B-chain) is a galac-
tose binding le~lin. The B chain is responsible for birding ricin to cell surfaces
by the i.Jeraciion wiih galactcsyl residues of membrane glycoproteins or glycolipids.
Thd E~chail? is the key to ihe cytotoxicity to ricin since it binds the A chain to
the target cells and suSsequently delivers the A chain into the cytosol. In the cytosol,
the A chain beir:g highly toxic, the penetraiion of a single toxin-molecule ;nay be
suHicient to cause cell death (113).
The mistletoe lectins are also of high biological activity. Three lectins have been
reponed trom mistletoe (114) ML I. ML \I, arld ML Ill. The mistletoe ledin 1 (ML
I) is a narurally occuring conjugate of an enzyme (A chain) and a ledin (B chain).
The important properties associated with the A chain are mitogeniciy and inhibition
of proteir~ synthesis. The B chain lectins activate macrophages and release lymphoki-
rles from lymphocytes (1 14). ML I is Dgaladose specific lectin, ML II D-galactose
i N-acetyl D-galactosamine specific and ML Ill. N-acetyl-D-galactosamine specific
lectin. The lectin fraction tram Viscum album has been repollad to be cytotoxic
to EAC cells, Sov 16 and SaAa m u s e ascites cells arid Zajdela hepatonla ascites
cells (1 15). Later, an immunotoxin consisting ot the enzymatically active A chain
of misiietoe lectin 1 and a mnoclonai antibodj against a surface protein on muse
leukemia L1210V cells was found to inhibit protein synlhesis in these cells (116).
rt has been proposed that the oiological activity of mistletoe preparations is due
to kIL I lectin (116. 117. 116).
1.2.6 Plants and plant products in cancer therapy
Traditional and folk medicines involving plant products have bng been used
for therapeutic purposes but scientific studies giving emphasis to theic mechanisms
of action have not made much progress. India is endowed with very rich flora and
we have our own lraditional system of medicine like Ayuweda a:d Siddha. A multi-
disciplinary approach is necessary to unearlh the therapeutic potectial of medicinal
pianjs.
During the investigation of the medicinal properties of the Periwinkle plant, Catha-
ranthus r o ~ ~ u s , it was found that certain extracts caused granulocytopenia in ani-
mals (1 7). an observation which subsequently led to the discovery d vinca alkaloids,
Vincristine and Vinblastine. These plant products act by inhibition of tubular assen-bly
and thus interfere with cell separation in mitosis (18). Both Vincristine and Vinblastine
are used in combination with bteomycin and cisplatin for the therapy of metastatic
testicular turnours (19). Vincristine's most important toxicity is a mixed motor-sensory
and automatic neuropathy (1 19), while that of Vinblastine is bonemarrow suppression
(118). Taxot was first isolated from the stem bark of the Western Yew, Taxus brevifo-
lia (14). Study of the natural and semisyntheti compounds of taxol has demonstrated
that an mtacl taxane ring and an ester side chain are required for cytotoxicity (15,
16). The cytotoxicity of taxol is probably related to micmlubule mediated interruption
of rnitosls
By using in vitro and in vivo screening procedures (12, 13) thousands of
planl extracts are tesled each year for their potential use as anticancer agents.
Antitumur principles have also been reported from Agrostitachys hookeri (120), Spo
rosperinum febr~tugum (121) Annona dsnsicoma (122) Isotheciurn subdiversiforme,
Thamnobryuni sandei (123), Polyfrichum obioense, Paroristolochia tlos-avis (124),
etc. and lrom many other plants. Amng the vast number of plants tested only
a very few numbers are found to be clinically useful. Active constituents of certain
plants effective against one or m r e of tumours, tested at Central D N ~ Research
Institute, Lucknow have been isolated, characterized and biologically evaluated.
These include Arnebin-1 from Amebia noblis , inhibited mouse leukemia and rat
walker carcinosarcoma (125). Celsioside, a saponin from Celsia ~ r ~ ~ ~ n d s l i a n a
has shown high activity in Walker carclnosarcoma system but Is toxic in the mouse
leukemia system (126). Antileukemic constituents fmm TiNMnia tagilillom (127) and
other blobgically active antiiumur agents from Coaxrlus pendulus, Roylea calycina
and Tephrosia candida have also been characterized (128). Other plants which
have shown varying degrees of antiiumour activity are Allium cepa (129), LifJ~St111m
neilgherrense (130), Semicarpus anacardium (131) and Rhazya stricta (132). Antiviral
and anli'ilmour activities of Cassia tistula, Aglai rox&rghiana and Zingiber Capita-
turn have been attributed to the presence of in:elfemn-like proteins in them (133).
Cytotoxic effed of different degrees and nature in mice has been reported in sesquiter-
pane lactone component of Parthenium hysterophorus and in Plumbagin from Plum
bago rosea.
On screening various spices and leafy vegetables for their influence on carcino-
gen detoxilying enzyme, glutathione - S-transferase (GST) in Swiss mice (134) it
was observed that Cuminum cyminum Linn (Cumin seeds), Papaver somniferum
Linn (Poppyseeds), Ferula asafoetida Linn (Asafoetida), Curcuma longa Linn. (Turme-
ric), Peer longum Linn. (Kandathipiii), Azadiracta indica Juss (Neem flowers), Sola-
num nigrum Linn (Manathakkali leaves), Moringa oleifera Lamk (Drurnstidc leaves),
Ociniu~n sanctum Linn (Basil leaves) are found to increase GST activity by more
than 78 par cent in the stomach, liver and oesophagus, high enough to be considered
as protective agents against carcinogenesis. There ars several reports about the
anlicarci:iogenic activity of sulfur containing compounds derived from garlic and onion
(135). Ellagic acid, a naturally occuring phenolic compound present in grapes, straw-
berries, rasp-benies and certain nuts (136, 137) is a well known chempreventive
agent against several chemical carcinogens (138). Eugirol, one cf the active ingre-
dients in cloves is reported to have antimutagenic action against several environmen-
tal mutagens (139). Certain spices ike black pepper, asafoetida, pippali and garlic
which are included in our diet have also been shown to be effective as inhibitors
of carcinogenesis i i~ animal mdels (140). Betal-leaf extract has been reported to
be an effective inhibitor of tobacco specific nitrosamineinduced cancers in experimen-
tal syscem (141).
Fro~n the foregoing paragraphs it can be seen that only a very few plant agents
are effectively being utilised for cancer therapy. The important plant products now
avsiiabia in the market are Vincristine, Vinblastine, VM 26 and VP 16. Some of
the plant dwgs are in the various stages of clinical trial and an intensive effort
is essential to bring many new plant dlugs to critical clinical evaluation. One of
the promising plants on which detailed investigations are now being carried out
and found to be useful in cancer therapy is European mistletoe and is reviewed
meticulously.
Plants of Loranthaceae famlly
The word mistletoe is applied to plants with a similar hemiparasitic life style
and a certain degree of taxonomical relationship in three families viz. Loranthaceae.
Viscaczae and Eremolegidaceae. With the exception of the genera nuytsia, atkinso-
Ilia and gaiadendron which parastte roots, they parasite aerial shoots of other higher
plants (142). For latier reason, !hey are also termed epiparasites. Because of their
special habit, mistletoes and especially European mistletoe have attracted special
interest in folklore and medicine throughou! the centuries. Taxonomic, morphological
snQ ptiysiological data on mistletoes have recently been described In detall (143).
A ra re detailed monqraphy on the biology, chemistrj and therapeutic action Of
this interesting plant has also beer: plblished (144). Mistletoes are hemiparaslic,
i.e. they depend for water am5 mineral nutrition on their respective hosts but are
able to produce cafbohydrates by photosynthesis. It has been shown that they con-
tain all pigments, chlorophyll a and b as well as carotinoids that are necessary
for photosynthesis (145). Through radiotracer experiments, it has been shown that
mistletoes have the same photosynthetic activity per unit leaf surface as the respec-
tive hcst plants (146). There is no significant transport of organic substances from
the host to tne parasite and vice versa. Similar resuns have been reported for the
American mistletou Phoradendron flavescens that belongs to the sarne family of
Viscaceae (147, 148). Earlier reports that, the Post largely influences the chemical
con~pu~nds of tha respactive m;stletoe, e.g., nlistletoe grown on pine trees p:oduces
pine-like :ignin and mistletce grown on hard wood trees produce hardwood-like lignirt.
could PO^ be proven (149, 150). kll mist:etoes including Viscum album usually havd
a h~ghc; mineral cantent lhafi the tost and especially the infected host branch
(151).
T t ~ e anticancer activity oi mistlztoe (Via-urn album) arid its alkaloids is vridely
docunierited (128). Since :he early lwentias, an aquecus extrac? of European mistle-
toe (V~scum album L) under tne trade name Iscador hss been used for the treatment
cf burr,-,- fiesplasi;? (152, 153) but controversial :eprts have also Seen appeared
5 153 dire lo the l jck of an acceptable scientific basis of care!cily controlled
clinicai trials. From a number of
lscador is beneficial in the post operative treatment of lung, colon. breast and cervical
carcinomas (153, 156 - 158). The anticancer activities of mistletoes have been and-
buted to a combination of cytotoxk (160 - 163) and immunological effects (158.
159. 163) and mistletoe treatments. unlike cytotoxic drugs, are not imrcunosuppres-
sive (153). Effect of a preparation from Visarm album (Iscador) on tumour devebp-
men: in vitro and in mice has been investigated (129) in detail and was lound
to be cytotoxic to animal tumour cells such as DLA and EAC jn viim and inh i ied
the growth of L-929 cells, CHO cells and KB cells at very Low concentrations.
Moreover, administration of lscador was lound to reduce asciles tumours and solid
tumurs produced by DLA cells and EAC cells. The effect of the drug could be
seer1 when tne drug was given either simultaneously alter the tumour development
or When glven prophylactically, indicating a mechanism of action very different from
other chemotherapeutic drugs. lscador was not found to be cytotoxic to
iy mphocyles
The pharmacological eflects of Viscum album (mistletoe) extracts used for can-
cer therapy are reported to be possibly due to water soluble poly~saccharUes of
the fresh plants and the fermented proprietary preparation lscador, which were isola-
ted and characterized ( I S ) , the main polysaccharide of the gr6en parts of Viscum
is a highly esteriliod galacturonan whereas Viscum berries contain a complex arabino-
galactan iMolecular weight 1,80,000 - 9,00,000). Immunological investigation indim-
ted :ha: none of tha carbohydrates showed significant stimulating effects on cells
of the u i i s~dc i f i ~ immune defense system. Ivlistletoe is reported !o be used in various
ways for medicinal purposes, as fermented preparation of the squeezed sap, Or
as alcoholic - aqueous extracts, sometimes in combination with other drug extracts
(164). Th6 main areas of application are (i) cardiovascular illness especially hypelten-
sion and arteriosclerosis (ii) cancer (iii) arthrosis. The three diluted mistletoe extracts
are lscador. Hel~xor and Plenosol are used for subcutaneous and intravenous applica-
tion. Although a large number of cor~stituents have aiready been isolated from mistle-
toe drug, 11 is not yet known which compounds are responsible for its reported
action. The flavonoids and phenol sarboxylic acids together with the phenylpmpanes
and lignans as well as the amines recently isolated (165, 166) are all possible agents
of cardiovascular activity. The presence of high molecular weight ~mpoutx ls , such
as lectins, viswtoxins and polysaccharides and in view of their cytotoxic (167) and
irnmunornodulatory properties (1 15) the lectins seemed to be the most interesting
of these three groups of constituents.
Mist!eloe ledins are reported to have high biological activity (1 14). The mistletoe
iectin - (Mil) is a naturally occuring conjugate of an enzyme (A chain) and a lectin
(B - chain). its cytotoxicity is caused by inhibiting the protein synthesis on the riboso-
mai level The prominent properties of the A chain are mitogenicity and inhibition
oi the protein synthesis in cell !ree syslenls. The A chair1 is also participating in
the iorrnat:on of im~nunotoxins. The B chain as well as ;he intact lectins activate
macrophayes and release lymphokines 1:om lymphocytes (168). Combination of selec-
tively cytotoric and immunopoter~tiating properties, mistletoe lectins and their chains
are decisive for the therapoiltic effects of mistletoe. The cytotoxicity of three mistletoe
leclins (MLI, MLIi and ?ilLiII) on a hcman tumoural cell line and ~tle relationship
between the primary structures of the N-terminal mistletoe lectin, A chains and that
of various .oxins with a related mechanism of action has been studied by Dietrich
e l a1 (1992) and reported that N-terminal sequences of the three A chains of MLI,
MLll and MLlll are identical and have interesting homology with the N-terminal sequen-
ces of the A chain of ricin like toxins and of single chain ribosome inhibiting proteins.
In addit~on, the three mistletoe lectins inhibit the growth of human tumour cell line
Molt-4, MLlll being the most potent, followed by MLll and MLI. This inhibition is
suppressed by addition of rabbl anti ML1 antibodies to the culured cells. The data
obtained suggest that the three ledins have amino acid sequences which show
extensive homology and exert very similar biological effects.
The lermented mistletoe preparation,lscador,inhibns the development of experi-
mental tumour cells (EAC cells, S-180 and Lewis lung carcinoma) so that the survival
time of treated animals is equal or higher than that of mice treated with 5-Flurouracil.
a well kcown ailtitunural agent (169). Moreover, ferrnenied lscador stimulates humo-
ral and celiular immunity in mice ( l W , 170). A marked increase in the weight of
the thymus, correspcnding to a higher proliferation rate of cortical thymocytes was
also observable and this effect was reversible (178). In cancer patients, fermented
lscador stimulates the activity of natural killer cells (171). The bacterially fermented
mislletoe preparation used in cancer therapy and the unfermented preparation have
been tested for its effect on rat hepatoma tissue culture (HTC) cells and human
leukemia Molt-4 cells and observed that unfermented lscador showed a much
stronger cytotoxic effect on these cells than that on HTC cells (172). While investiga-
ting the growth inhibitory effect of lscador on a variety of tumur cells (189, 173-175).
mutt, evidence have accumulated to suggest that lscador nlaY Flay a mle in
modulation, More than 60 years ago it was considered possible (176) that Iscador
had an immunotherapeutic importance for cancer, but the first significant impulse
for the recent immunological research was given 15 years ago by Nienhaus and
Leroi (174) and Nienhaus s t &I (177). They demonstrated that repeated intraperito-
neal or intravenous injections of lscador in mice induced an enlargement of thymus.
Later, Rerilea gt (178) confirmed and elaborated these resuns. They showed
in CG-l rnice that the thymic trophii ir~iluence was mainiained over a long period
of time following repeated intraperitoneal injections of lscador and that this resulted
froni an increased proliferation of cortical thymocytes. The quality of this proliferative
activity diltered from the reactions observed in autoimmune disease and following
direct antlgen injection. Thyrnocytes of iscador-treated animals were significantly
more responsive to concanavalin A (Con A! than those of untreated controls. Blok-
sma cl a! (1 63, 170) and kwaja gl (1 62) have found that Viscum album prepara-
tions may be a good adjuvant for delayed hypersenslivity response to sheep red
blood cells (SRBC) in mice. Rentea at a1 (178) and Bloksma 81 A (163, 170) have
demonstrated that lscador augmented the amount of antibody produced by animals
injected with SRBC. Bloksma 81 (170) have investigated the effect of Iscador
on non-s;:?;ific inflammation with the foot pad swelling test in mice. It is not yet
clear, wh~ch pafi of the immune response plays a dominant role in the development
01 resistance to tunlour growth. It is now generally accepted that the slightly immuno-
genic lu:rwur associated antigens usually fail to induce a specific immune response
(779 - 182).
One ol the most investigated role in this spontaneous system is that of natural
Killer (NU) cells. Convincing evidence have documented that NK-cells may play
a role In lnhiblting tumour growth (183 - 187) and an inverse correlation has been
reported between NK status and metastasis (188 - 191). Allhough the evidence
Is strongly In favour of NK-cell participation in natural host defense mechanism,
it is apparent that natural resistance is probably a heterogenous phenomenon gover-
ned by the complex interaction between diiferent cell types and the products there
in. Convinc~ng evidence have acarmulated showing that neutrophil granulocytes (192
- 194) and monocyles/macrophages (195 - 197) can play an important role in resistan-
ce to tumour groLYth and metastasis &I _oh. It is quite likely that in varying situations
different etfector cells may be the important fador involved in the tumur defense.
The irnmunomodulatory effect of lscador have been investigated in detail by Hajto
(1986) (198) and concluded that the significant allerations in various immunological
parameters (NWADCC activity, LGL frequencies, mitogen responses to PHA and
ConA, chemiluniinescence response of granulocytes during phagocytosis, non-
specific cytotoxic activity of macrophages) confirmed that lscador rnay have an
important I de in the immunomodulatory therapy of cancer.
Further investigation on the immunomodulatory activity of a peptide isolated
from V~scum album extract show (31) an increased natural Killer cell activity (NK
act~vity) i r i the normal animals and tumour bearing animals on administration ol
the peptide at the rate of 2 uq'ml. Pdministration of the peptide also stimulated
antibody oependant cellular cytotoxicity (ADCC) which was expressed maximally on
the 4th day There was also an increase in antibody fcrming cells in the spleen,
and an!oixdy titer was increased in the animals treated with the peptide. Activity
of the crude plant extract coincided with the activity of the peptide, indicating that
the isolated peptide is mainly responsible for the immunostimlatory activity present
in V~scum album extract.
Possible tumour reducing mechanism of a peptide of approximate m o l 8 ~ ~ k i r
weight 5000 isolated from mistletoe extract (Iscador) has been studied by Kuttan
et &, (1330) (29) and observed that this Isolated materiai at a concentra'on of -
1 0 - 7 ~ was found to produce 100 per cent cylotoxiciiy ta KB cells, CHO cells, LB
cells, DLA cells and EAC cells. At a concenll.alion of 2.5 mpkg body w e g ~ , this
rnater~al reduced the growth of sclid tumurs induced in mice by DLA Cells. The
turnour reducing action o: the isolated material couid be seen when ihe drug was
given either along with the cells after tumour development or when given prophylacti-
cally. A receptor for this peptide was found in the cell sonicates of DLA cells as
well as in the ascites fluid which inhibited the cytotoxicity of the isolated component.
tiowzver. ;he receptor was not seen in the cell sonicates of lymphocytes which
are i:oi sdsceptibls to the cyiotoxic action of the isolated component indicating that
the b~rldirig of the active ingredient in the lscador to :he cell surface receptor is
rleeoed for its action. Isolated peptide from !he Viscum album extract in cellular
infilteration and subsequent turnour necrosis has been demonstrated both in vitro
and In vivo by Kuttan et a1 (200). Spleen cells from animals treated w lh a very
smal! quantity of this peptide viere fcunc! to have increased response to PHA and
ConA indicating that more mature lymphocytes are produced at the peptids administra-
tion. Moreover injection of the peptide at the lesion site produced infiltration of lympho-
cytes and macrophages and finally producilyl necrosis of the tumur. This peptie
also stimulated macrophages in vitro and in vivo and activated macrophages were
found to have cytotoxic activity towards 1-929 fibroblasts.
Antitumorous and immunomodulatory eflects of the Viswm album L preparations
lsorel was studied by Jurin 6 gj (1993) (201) and found that in mice, an increased
number ot plaque forming cells to SRBC followed consequent on the injection of
lsorei together with SRBC. Further survival time of a foreign skin graft was shortened
if lsorel was applied at the correct time. Finally, suppressed immune reactivity in
tumurous mice recovered following lsorel injection. lsorel was furlher shown to
be cytotoxic to turnour cells in vitro. Its application to tumour bearing mice wuM
prolong their lives but without any therapeutic effect. However, a combination of
local irradiation and lsorel was very eflective : following 43 Gy of local irradiation
to a transplanted methyl cholanthrene - induced fbrosarcoma (Volume 240 mrn3
) growing in syngeneic CBAiHZgr mice, the tumour disappeared in about 25 per
cent of the animals, the addition of lsorel increased the incidence of cured animals
to 65 per cent. The combined action of Isorel, influencing tumour viability on the
one hand and the host's immune reactivity on the other, seems to be favourable
for ils anli!.mour action in vivo. Macrophages from mice treated with Viscum album
extract mere shown lo be active in inhibiting the pro!iferation of turnour cells in
culture (292). These activated rnacrophages have now been shown to protect mice
from dying of progressive turnours when injected intraperitoneally into the mice. Pro-
phylactic as well as multiple treatmt?nts with macrophages activated with Viscum
album extracts seemed more eflective than a single treatment. This findings suggest
that in addition to a direct cytotoxic effect of Viscum album extract the activation
ot rnacropliagos rrlay corllribulo to tllo ovcrall arltitulrlour actlvity ot tlio drug.
Reduction of leukopenia in mice by Viscum album administration during radiation
and chemotherapy have been reported by Kuttan et a1 (1993) in that Viscum album
extract was found to reduce the leukocytopenia produced by radiation and cyclophos-
phamide treatment in animals (203). Weight loss due to radiation was considerably
reduced by Viscum album extract whereas weight loss due to cyclophosphamide
was not altered. Haemoglobin levels also were not aftected by Viscum album extract
administration. The results indicated that Viscum album extract reduces lymphocyto-
penia and hence could be used along with chemotherapy and radiation therapy.
Increasing concentration of Viscum album L extracts were shown to significantly
reduce sister chromatid exchange (SCE) frequency of PHA - stimulated peripheral
blood morionuclear cells (PBMC) of healthy individuals (205). Purified lectin from
Viseurn album was shown to induce a release of tumour necrosis factor (TNF
- a) (116, 206) and of inter leukin-1 (IL-I) and interleukin-6 (116) and of interferon
r (IFN-r) by PBMC. The cytokines IL-2, IFN-r and TNF have been shown to increase
cell proliferation and SCE (207 - 210), whereas IFN-a and IFN-B (211, 212) reduced
SCE frequencies. Therefore. the DNA protective etfect of the V;scum album L prepara-
tion may have some similarities with the lipopolysaccharide induced radioprotection
of immune cells (213), i.e, treatment with Wscum album L extracts could be the
initial step in a cascade of sequentially interacting events or substances (cytokines),
leading to DNA protection and SCE reduction.
Interaction between Viscum album and DNA was studied (214) by amplification
of specific human pepsinogen A gene promoter sequences by polymerase chain
reaction (PCR). It was shown that promoter sequences incubated with Iscador beca-
me insensitive to methylation by specific DNA methyl transferases by destruction
of DNA methyl transferase activity. @ussing a 1995 (215) determined the SCE
frequency and the proliferation index of amniotic fluid cells after addition of increasing
concentrations of Viscum album L extracts. The Brd U - induced SCE frequency
of AFC re:( ained stable only at a low Viscum album L concentration of 20 uglml,
a drug concentration, which related to body weight and body fluid corresponds approxi-
mately to that dose which cancer patients require daily by subartaneous injections
(216).
HELICANTHES ELASTICA (Famlly - Loranthaceae)
Helicanthes elastica is a dichotomously branched shrubby parasite, leaves
ovate or elliptic, three to five nerved, with obtuse tip. Flowers are sessile clustered
at the nodes. Calyx tube is ellipsoMal in shape and the limbs are cylindrical. Corolla
lobes are about 3 cm long. Fruit is a subglobose or avoid berry.
Helicanthes elastica grown on host trea Mangifera M i c a is shown in fig. 1.1.
So !ar no i<> rk regarding the tnerapeutic potential of Helicanthes elastica has been
reported earlier.