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.