Antioxidant and Antitumor Activities on Catunaregum spinosa

1. ~ 464 ~
_______________________________________
* Corresponding author: A.Chitra
E-mail address: chitrampharm@gmail.com
Available online at www.ijrpp.com
Print ISSN: 2278 – 2648
Online ISSN: 2278 - 2656 IJRPP | Volume 2 | Issue 3 | 2013 Research article
Antioxidant and Antitumor Activities on Catunaregum spinosa.
*A.Chitra1,2
, N.Senthilkumar2
, M.Asraf ali1
1
Sunrise Univercity, Alwar, India.
2
JKKMMRF’s College of Pharmacy, B. Komarapalayam, Tamilnadu, India
ABSTRACT
The methanol extract of Catunaregum spinosa (MECS) were evaluated for antitumor activity and antioxidant
activity against Dalton’s ascites lymphoma (DAL) bearing Swiss albino mice. The extract was administered at
the doses 200 and 400mg/kg body weight per day for 14 days after 24h of tumor inoculation. After the last dose
and 18 h fasting, the mice were sacrificed. The present study deals with the effect of MECS on the growth of
transplantable murine tumor, life span of DAL-bearing hosts, hematological profile, biochemical and
antioxidant profile. MECS caused significant decrease in tumor volume, packed cell volume, and viable cell
count; and it prolonged the life span of DAL-tumor bearing mice. Hematological profile converted to more or
less normal levels in extract-treated mice. The lipid peroxidation was increased in tumor bearing animals, after
treatment with MECS antioxidant levels increased significantly. The results indicate that MECS exhibited
significant antitumor activity in DAL-bearing mice.
KEYWORDS: Catunaregum spinosa, Transplantable tumors, Anticancer, Dalton’s ascites lymphoma.
INTRODUCTION
Cancer is the uncontrolled growth of abnormal
cells in the body. This results from a series of
molecular events that fundamentally alter the
normal properties of cells. In cancer cell the
normal control systems that prevent cell
overgrowth and the invasion of other tissues are
disabled. These altered cells divide and grow,
display uncontrolled growth, invasion and
sometimes metastasis.
Experimental tumor models have a wide role in
anticancer drug discovery. A Dalton’s ascites
lymphoma (DAL) tumorigenesis model in Swiss
albino mice provides a convenient model system to
study antitumor activity within a short time.
Following transplantation of DAL cells into the
abdominal cavity of healthy recipient mice,
tumorigenesis begins immediately and
aggressively. Free radicals are the chemical
species contains at least one ‘unpaired of electron’.
Reactive oxygen species (ROS) are the free
radicals associated with the oxygen atom or their
equivalents and have stronger reactivity with other
molecules than with molecular oxygen [O2].
Free radicals are implicated in many pathological
conditions by irreversible damaging the structure of
biological molecules like cell membranes, DNA,
Proteins etc. these free radicals can directly
interacting with DNA produce single or double
strand DNA breaks, DNA cross linking, purine,
pyrimidine, or deoxyribose modifications, and
DNA cross-links. Persistent DNA damage can
result in either arrest or induction of transcription,
induction of single transduction pathways,
replication errors, and genomic instability, all of
which to cancer. [Marian et al., 2004].Plants are
the rich source of medicines from ages. They
produce bioactive molecules which can used to
ameliorate various types of disorders. Over the last
few decades there has been increased interest by
pharmaceutical industries to discover the new
drugs from the ethno botanical provide new and
alternative drugs to synthetic drugs for treatment of
International Journal of Research in
Pharmacology & Pharmacotherapeutics

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dreadful diseases. Potent anticancer drugs like
taxol, vinblastine, vincristine, the camptothecin
derivatives, topotecan and irinotecan, and etoposide
derived from plant sources and they are in efficient
clinical use. Catunaregum spinosa (Family:
Rubiaceae) is a rigid shrub or tree, upto 9 m in
height and 1.2 m in girth found throughout India up
to an altitude of 1900 m. The species is common as
under growth in the sub Himalayan track and
elsewhere. The flowers and fruits are edible; the
flowers are eaten as a vegetable in Nasik district.
The raw fruits have a highly astringent taste due to
high tannin content. The fresh fruits contain high
amount of carbohydrates and saponins. The seed
contains essential oil and organic acid. The main
use of Catunaregum spinosa is nauseant,
expectorant, anthelmintic and abortifacient
properties. It also showed hypoglycemic,
insecticidal and anti cancer activities. The seeds are
tonic to induce appetite. Their decoction is taken
for relieve from headache. The bark is used to
diarrhea and dysentery.
Experimental animals
Male Swiss albino mice (20-25 g) were used for the
experimental study. The animals were maintained
under standard condition in polyprophylene cage
and provided with food and water ad libitum. The
animals were kept on fasting overnight prior to the
experimentation and all the procedure used in these
studies were approved by the institutional Animal
Ethics Committee.
Tumor cells
DAL, cells were obtained from Amala Cancer
Institute, Thrissur, Kerala, India. The DAL cells
were maintained by intraperitoneally inoculation of
2×106
. Ascetic fluid was drawn out from DAL,
tumor bearing mouse at the log phase (days 10-12
of tumor bearing) of the tumor cells. Each animal
received 0.2ml of tumor cell suspension containing
2×106
tumor cells intraperitioneally.
Treatment schedule
60 Male Swiss albino mice were divided into five
groups (n=10) and given food and water ad libitum.
All the animal in each groups except group-I
received DAL cells (2×106
cells/mouse i.p). This
was taken as day ‘0’. Group-I served as normal
control (25% Tween 80 per oral) and group-II
served as DAL control. 24 h after DAL
transplantation,
Grop-III and group IV received methanol extract of
Catunaregum spinosa at a dose of 200 and 400
mg/kg/oral for 14 consecutive days, respectively.
Group-V received reference drugs 5-fluorouracil
for 14 consecutive days. 24 hours of last dose, 5
animals of each group were sacrificed to study the
tumor growth parameters (mean survival time,
viable, non-viable cell, tumor volume, tumor
weight and tumor packed cell volume), antioxidant
and hematological parameters and the rest were
kept with food and water ad libitum to check
percentage increase in life span of the tumor host.
In Vitro-cytotoxicity study
DLA cells (1×106
) in phosphate buffer saline (PBS)
and different concentrations (50, 100, 200, 400,
600, 800, 1000, 1600 µg/ml) of MECS were
incubated at 370
C for 3 hrs in 5% CO2 atmosphere
in the filtered cap, flat bottom cell culture flasks.
The viability of cells was determined by Trypan
Blue dye exclusion method.
No.of Dead cells
% cell viability = X 100
No.of viable cells + No.of dead cells
Tumor growth parameters
Tumor volume and weight
After 14 days of treatment, mice were dissected
and the ascetic fluid was collected from peritoneal
cavity. The volume was measured by taking it in a
centrifuge tube and weighed immediately.
Viable and non-viable tumor cell count
The viability and nonviability of the cell were
checked by trypan blue dye exclusion assay. The
cells were stained with trypan blue (0.4% in normal
saline) dye. Live (viable) cells actively pump out
the dye by efflux mechanism where as dead (non-
viable) cells do not. The number of viable and
nonviable cells was counted [Bala et al., 2010].
Number of cells x dilution factor
Cell count =
Area x thickness of liquid film
Tumor packed cell volume

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The ascetic fluid was collected into Wintrobe’s
tube and it was centrifuged at the rate of 3000 rpm
for a period of one hour. The volume of packed
cells read directly as percentage.
Percentage increased in life span
The effect of MECS on percentage increases in life
span was calculated on the basis of mortality of the
experimental mice.
Mean survival time of
the treated group
Increased
life span(%) = X 100
Mean survival time of the
control group
First death + last death
Mean survival time* =
2
*Time denoted by days.
Hematological parameters
At the end of the experimental period, blood was
collected from retrorbitalpluxes and used for the
estimation of hemoglobin (Hb) content, red blood
cell (RBC) count, white blood cell (WBC) count,
packed cell volume (PCV) and differential count by
standard procedures.
Biochemical parameters
The remaining blood was centrifuged and serum
was used for the estimation of liver biochemical
parameters like Serum glutamic pyruvic
transaminase (SGPT), Serum glutamic oxaloacetic
transaminase (SGOT), Albumin, Total protein
(TP).
Antioxidant activity
The liver was excised, rinsed in ice cold normal
saline followed by cold 0.15M Tris-Hcl (pH 7.4),
blotted and weighed. The homogenate was
processed for estimation of Lipid peroxidation
(LPO), Reduced glutathione (GSH), Glutathione
peroxidase (GPX), Glutathione-S-Transferase
(GST).
Effect of on normal peritoneal cell count
To evaluate effect of MECS on normal peritoneal
cells, 3 groups of normal mice (n=4) were taken.
One group was treated with 400 mg/kg p.o. of
MECS and the second group received the same
treatment for 2 consecutive days. The untreated
third group was used as control. Peritoneal exudate
cells were collected after 24 h treatment by
repeated intraperitoneal wash with normal saline
and counted in each of the treated groups and
compared with those of the untreated group.
Effected of on solid tumor
Mice were divided into two groups (n = 4). DAL
cell lines (1x106
cells/mice) were injected into right
hind limb (thigh) of all mice intramuscularly. The
group I used as DAL tumor control. The group II
treated with MECS 400 mg/kg/oral for 14 days.
Tumor mass was measured form 15th
day of tumor
induction. The measurement was carried out every
5th
day for a period of 30 days. Tumor mass
volume was measurement using following formula
V=4/3 r2
, where r is the mean of r1 and r2 which
are two independent radii of the tumor mass.
Statistical analysis
All data are expressed as mean ± S.E.M. Statistical
significance (p) calculated by ANOVA followed by
Dennett’s (tumor volume, tumor weight, viable,
non viable, mean survival time, tumor PCV) and
Benferroni tests (hematological, SGPT, SGOT,
Total Protein, albumin, Antioxidant parameters).
P< 0.05 was considered as statistically significant.
RESULTS
In Vitro - cytotoxicity study
The In Vitro cytotoxicity effect of MECS at
various concentration 50, 100, 200, 400, 600, 800,
1000, 1600 µg/ml on DAL cell lines using tryphan
blue dye exclusion assay method has to be done.
The percentage of cell viability 10%, 25%, 30%,
42%, 54%, 70%, 85%, 92% respectively. The
IC50 value was found to be 1000 µg/ml.
Effect of MECS on mean survival time
The effect of MECS on mean survival time were
shown in table 1, on oral treatment of MECS to the
tumor induced DAL mice, the mean survival time
of DAL control group was found to be 22 ± 0.75,
while it increased to 29 ± 0.25 (MECS 400 mg/kg)
respectively in MECS treated groups and whereas
the standard drug 5-fluorouracil (20 mg/kg) treated
group had a mean survival time 31± 0.21.
Effect of MECS on tumor growth
The effect of MECS on tumor growth response
were shown in table 1, after treatment with MECS
(200 and 400 mg/kg) reduced the tumor volume,
viable tumor cell count and tumor packed cell

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volume in a dose-dependent manner as compared
to that of the DAL control group. Furthermore,
nonviable tumor cell counts at different doses of
MECS were significantly increases in a dose-
dependent manner.
Effect of MECS on hematological
parameters
The effects of MECV on hematological parameters
were shown in table 2, Hemoglobin content and
RBC count in the DAL control group was
decreased as compared to the normal control group.
After treatment with MECS at various doses 200
mg/kg, 400 mg/kg significantly increases the
hemoglobin content and RBC count and brought up
to the near or less to the normal levels. The total
WBC count was found to be increased significantly
in the DAL control group when compared with the
normal group. Administration of MECS in DAL –
bearing mice siginificantly reduced the WBC count
as compared with the DAL group.
Effect of MECS on biochemical parameters
As shown in table 3, SGOT, SGPT, albumin levels
were increased significantly and total protein levels
were decreased as when DAL control group
compared to the normal control group. After
treatment with MECV at the dose of 200 mg/kg,
400mg/kg and FU significantly decreased the
elevated SGOT, SGPT, albumin to normal levels
and increased total protein levels.
Effect of MECS on antioxidant activity
As shown in table 4, in the DAL, group, the LPO
level was increased and SOD, CAT, reduced GSH,
GPx, GST levels were decreased significantly as
compared to the normal control. After treatment
with MECS at different doses (200 mg/kg, 400
mg/kg) and 5-FU significantly decreases the LPO
and increased the SOD, CAT, reduced GSH, GPx,
GST levels.
Effect of MECS on solid tumor growth
There was reduction in the tumor volume of mice
treated with MECS 400mg/kg from 15th
day to 30th
day. On 30th
day tumor volume of DAL control
animals was 7.3±1.17 ml, whereas for the extract-
treated group it was found to be 0.05±0.24 ml as
shown in table 5.
Table 1: Effect of methanol extract of Catunaregum spinosa tumor growth parameters
Parameters DAL DAL + MECS
200 mg/kg
DAL + MECS
400 mg/kg
DAL + 5- FU
20 mg/kg
Mean survival time (days) 22± 0.75 25± 0.32 29± 0.25 31± 0.21
Increased life span (%) - 24.7 31.2 42.9
Tumor volume (ml) 4.28± 0.06 3.26± 0.04 2.16± 0.02 2.34± 0.04
Tumor packed cell volume (ml) 2.11 ± 0.05 1.08 ± 0.03 0.84 ± 0.02 0.63 ± 0.02
Viable cell count (x107
cells/ml) 12.8 ± 0.07 9.42 ± 0.06 6.21 ± 0.04 5.3 ± 0.05
Non viable cell count (x107
cells/ml) 0.45 ± 0.03 0.76 ± 0.06 0.92 ± 0.05 0.87 ± 0.06
Data are expressed at the mean ± SEM. N=5. P<0.01, extract treated groups compared
with the DAL group.
Table 2: Effect of methanol extract of Catunaregum spinosa on hematological parameters
Parameters Control DAL
DAL + MECS
200 mg/kg
DAL + MECS 400
mg/kg
DAL + 5 FU
20 mg/kg
Hemoglobin 14.3± 1.12 7.2 ± 0.32 8.9 ± 0.49 10.4 ± 0.6* 10.2 ± 0.9*
RBC(x106
cell/mm3
) 12.2 ± 0.61 7.6 ± 0.5 8.5 ± 0.5 10.5 ± 0.4 12.2 ± 0.6
WBC(x104
cell/mm3
) 0.61 ± 0.12 5.41 ± 0.5 3.45 ± 0.32* 1.4 ± 0.12 1.54 ± 0.16*
Polymorph (%) 21.2 ± 1.26 66.7 ± 1.15 49.4 ± 1.42 38.5 ± 1.34 38.2 ± 1.24
Lymphocyte (%) 79.6 ± 2.23 34.4 ± 1.72 51.3 ± 1.11 60.2 ± 1.63 58.7 ± 1.82
Data are expressed as the mean ± SEM, extract treated group was compared with DAL
group. P< 0.01, *P< 0.05.

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Table 3: Effect of methanol extract of Catunaregum spinosa on biochemical parameters
Parameters Control DAL DAL+MECS
200 mg/kg
DAL+MECS
400 mg/kg
DAL+5FU
20 mg/kg
SGPT(U/L) 20.7± 1.4 48.6± 2.3 36.8± 1.3 31.7± 1.12 31.2± 1.3
SGOT(U/L) 121.6± 22.3 175.8± 17.3 155.4± 7.9 121.5± 3.4 87.5± 2.9
Albumin (gm %) 2.3± 0.7 12.5± 0.5 3.8±0.81 2.7± 0.4 2.5± 0.12
Total protein (gm %) 5.81± 0.64 2.9± 0.42 3.71± 0.92 4.59± 0.83 6.2± 0.66
Data are expressed as the mean ± SEM, extract treated group was compared with DAL group. P< 0.01.
Table 4: Effect of methanol extract of Catunaregum spinosa on antioxidant parameters
Parameters Control DAL DAL+MECS
200 mg/kg
DAL+MECS
400 mg/kg
DAL+5FU
20 mg/kg
LPO (ng of MDA/mg
protein)
0.31± 0.02 2.4± 0.48 0.82± 0.08 1.43± 0.15 1.56± 0.12
SOD (U/mg protein) 0.22± 0.06 0.02± 0.01 0.08± 0.002 0.14± 0.03 0.16± 0.02
CAT (U/mg protein) 0.95± 0.11 0.15± 0.04 0.42± 0.02 0.38± 0.13 0.77± 0.07
GSH (mg/g wet tissue) 0.76± 0.14 0.33± 0.09 0.48± 0.17 0.58± 0.15 0.59± 0.17
GPx (U/mg protein) 0.03± 0.002 0.0013±
0.002
0.005±
0.0003
0.016± 0.001 0.018±0.003
GST (U/mg protein) 0.026±0.003 0.007±0.0004 0.018±0.000 0.016±0.004 0.019±0.005
Data are expressed as the mean ± SEM, extract treated group was compared with DAL group. P< 0.01.
Table 5: Effect of methanol extract of Catunaregum spinosa on solid tumor growth
Solid tumor volume in ml
Groups 15th
day 20th
day 25th
day 30th
day
DAL control 0.086±0.01 2.6±0.5 4.0±0.7 7.3±1.17
MECS 400 mg/kg 0.25±0.05 0.19±0.18 0.09±0.09* 0.05±0.24*
Data are expressed as the mean ± SEM, extract treated group was compared with DAL group. P<0.01, *P<0.001.
DISCUSSION
The present study was carried out to evaluate the
antitumor effect of MECS in DAL bearing mice.
The MECS treated animals at the doses of 200 and
400 mg/kg significantly inhibited the tumor
volume, packed cell volume, tumor cell count, and
brought back the hematological parameters to more
or less normal levels. In DAL bearing mice, a
regular rapid increase in ascities tumor volume was
noted. Ascited fluid is the direct nutritional source
for tumor cells and a rapid increase in ascites fluid
with tumor growth would be a means to meet the
nutritional requirement of tumor cells. Treatment
with MECS increased the percentage of tryphan
blue positive stained dead cells in tumor bearing
mice. The reliable criteria for judging the value of
any anticancer drug are the prolongation of the life
span of animals. The MECS decreased the ascited
fluid volume, viable cell count, and increased the
percentage of life span. It may be concluded that
MECS decreasing the nutritional fluid volume and
arresting the tumor growth, this could be the reason
for the increased the percentage of life span of
DAL bearing mice. Usually, in cancer
chemotherapy the major problems that are being
encountered are of myelosuppression and anemia.
The anemia encountered in tumor bearing mice is
mainly due to reducdtion in RBC or hemoglobin
percentage, and this may occur either due to iron
deficiency or due to hemolytic or
myelopathicconditions. After the repeated
treatment, MECS able to reverse the changes in

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hematological parameters hemoglobin content,
RBC, and WBC counts near to normal levels. This
indicated that MECS is showing protective action
on the hemopoietic system.
The generation of free radicals in vivo is a constant
phenomenon due either to physiological
metabolism or pathological alterations. These
generated free radicals are the main cause of lipid
peroxidation which is an autocatalytic free radicals
chain propagating reaction, is known to be
associated with pathological conditions of a cell.
The oxidation of proteins, lipids, nucleotides and
carbohydrate causes chemical modification, leads
to damage of above. Malondialdehyde is the end
product of lipid peroxidation, was reported to be
non cancerous cells. Cells have developed
enzymatic systems (antioxidant enzymes) like
SOD, catalase and glutathione which convert
oxidants into non-toxic molecules, thus protecting
the organism from the deleterious effects of
oxidative stress. Glutathione (a trepeptide), usually
the most prevalent intracellular thiol, functions
directly or indirectly in a variety of cellular
processes. Reduced glutathione plays an important
role in defense mechanisms by acting as an
antioxidant or by reacting with electrophiles and
toxic agent to form conjugated that are eliminated
from the cell. SOD, CAT, and glutathione
peroxides are involved in the clearance of
superoxide and hydrogen peroxide. SOD catalyses
the diminution of superoxide, which has to be
eliminated by glutathione peroxidase and/or
catalase. The inhibition of SOD and CAT activities
as a result of tumor growth was also reported.
Similar finding were observed in the DAL bearing
drug treated mice. As above stated like the
decreased levels of SOD, Catalase and GPx levels
were observed in the present study, after drug
treatment with different doses the levels of these
enzymes were significantly increased.
Natural antioxidants are playing a great role in free
radicals scavenging activity. Some tritepenoids and
flavonoids are found to have anticancer and
antioxidant activity. MECS shows the presence of
triterpene and flavonoids which may act as
anticancer and antioxidant principles in the
diseased condition. In our earlier studies, we found
that MECS posses hepatoprotective and antioxidant
properties. The free radicals hypothesis supported
the fact that the antioixidants effectively inhibit the
tumor, and the observed properties may be
attributed to the antioxidant and antitumor
principles present in the extract.
CONCLUSION
The present study demonstrated that MECS
increased the life span of DAL tumor bearing mice.
And decreased the lipid peroxidation and thereby
augmented the endogenous enzymes in the liver.
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