Munson, L.S. Harris, M.A. Friedman, W.L. Dewey, and R.A. Carchman
of the National Cancer Institute, Vol. 55, No. 3, September 1975
by Public Health Service grant DA00490 from the National Institute on Drug Abuse,
Health Services & Mental Health Administration; by a grant from the Alexander
and Margaret Stewart Trust Fund; and by an institutional grant from the American
of Pharmacology and the MCV/VCU Cancer Center, Medical College of Virginia, Virginia
Commonwealth University. Richmond, Va. 23298
lung adenocarcinoma growth was retarded by the oral administration of delta-9-tetrahydrocannabinol,
delta-8-tetrahydrocannabinol, and cannabinol (CBN), but not cannabidiol (CBD).
Animals treated for 10 consecutive days with delta-9-THC, beginning the day after
tumor implantation, demonstrated a dose-dependent action of retarded tumor growth.
Mice treated for 20 consecutive days with delta-8-THC and CBN had reduced primary
tumor size. CBD showed no inhibitory effect on tumor growth at 14, 21, or 28 days.
Delta-9-THC, delta-8-THC, and CBN increased the mean survival time (36% at 100
mg/kg, 25% at 200 mg/kg, and 27% at 50 mg/kg;, respectively), whereas CBD did
not. Delta-9-THC administered orally daily until death in doses of 50, 100, or
200 mg/kg did not increase the life-spans of (C57BL/6 X DBA/2) F (BDF) mice hosting
the L1210 murine leukemia. However, delta-9-THC administered daily for 10 days
significantly inhibited Friend leukemia virus-induced splenomegaly by 71% at 200
mg/kg as compared to 90.2% for actinomycin D. Experiments with bone marrow and
isolated Lewis lung cells incubated in vitro with delta-8-THC and delta-9-THC
showed a dose-dependent (10 -4 10 -7) inhibition (80-20%, respectively) of tritiated
thymidine and 14C -uridine uptake into these cells. CBD was active only in high
concentrations (10 -4)
into the physiologic processes affected by the psychoactive constitutuents of
marihuana [delta-9-tetrahydrocannabinol (delta-9-THC) and delta-8-tetrahydrocannabinol
(delta-8-THC)] purified from Cannabis sativa are extensive (1). However, only
recently have attempts been made to elucidate the biochemical basis for their
cytotoxic or cytostatic activity. Leuchtenberger et al. (2) demonstrated that
human lung cultures exposed to marihuana smoke showed alterations in DNA synthesis,
with the appearance of anaphase bridges. Zimmerman and McClean (3), studying macromolecular
synthesis in Tetrahymena, indicated that very low concentrations of delta-9-THC
inhibited RNA, DNA, and protein synthesis and produced cytolysis. Stenchever et
al. (4) showed an increase in the number of damaged or broken chromosomes in chronic
users of marihuana. Delta-9-THC administered iv inhibited bone marrow leukopoieses
(5), and Kolodny et al. (6) reported that marihuana; may impair testosterone secretion
Furthermore, Nahas et al. (7) showed that in chronic marihuana users there is
a decreased lymphocyte reactivity to mitogens as measured by thymidine uptake.
These and other (8) observations suggest that marihuana (delta-9-THC) interferes
with vital cell biochemical processes, though no definite mechanism has yet been
established. A preliminary report from this laboratory (9) indicated that the
ability of delta-9-THC to interfere with normal cell functions might prove efficacious
against neoplasms. This report represents an effort to test various cannabinoids
in several in vivo and in vitro tumor systems to determine the kinds of tumors
that are sensitive to these compounds and reveal their possible biochemical sites
tumor systems used were the Lewis lung adenocarcinoma, leukemia L1210, and B-tropic
lung tumor: For the maintenance of the Lewis lung carcinoma, approximately 1-mm3
pieces of tumor were transplanted into C57BL/6 mice with a 15-gauge trocar. In
experiments involving chemotherapy, 14- to 18-day-old tumors were excised, cleared
of debris and necrotic tissue, and cut into small fragments (=1mm3). Tumor tissue
was then placed in 0.25% trypsin in Dulbecco's medium with 100 U Penicillin/ml
and 100 mcg streptomycin/ml. After 90 minutes' incubation at 22 Degrees C, trypsin
action was stopped by the addition of complete medium containing heat-inactivated
fetal calf serum (final concentration, 20%). Cells were washed two times in complete
medium, enumerated in a Coulter counter (Model ZB1) or on a hemocytometer, and
suspended in serum-free medium at a concentration of 5 X 10 6 cells / ml. Next
1 X 10 6 cells were injected into the right hind gluteur muscle, and drugs administered
as described in "Results." Standard regimens provided for 10 consecutive
daily doses beginning 24 hours after tumor inoculation. Body weights were recorded
before tumor inoculation and weekly for 2 weeks. Tumor size was measured weekly
for the duration of the experiment and converted to mg tumor weight, as described
by Mayo (10).
leukemia: B-tropic Friend leukemia virus (FLV) was maintained in BALB / c mice,
and drug evaluation performed in the same animals. Pools of virus were prepared
from the plasma of mice given FLV and stored at -70 Degrees C. In experiments
with FLV, 0.2 ml of a 1/20 dilution of plasma (derived from FLV-infected mice)
in medium was inoculated ip into BALB / c mice. Cannabinoids were administered
orally daily for 10 consecutive days beginning 24 hours after virus inoculation.
Twenty-four hours after the last drug administration, the mice were killed by
cervical dislocation, and the spleens removed and weighed. Mice not given FLV
were treated as described above, to evaluate possible drug-induced splenomegaly.
leukemia: The murine leukemia L1210 was maintained in DBA/2 mice by weekly transfers
of 10 (to the fifth power) cells derived from the peritoneal cavity. In these
experiments, 10 (fifth power) leukemia cells were inoculated ip into (C57BL/6
X DBA/2) F 1 (BDF 1) mice, and the mice were treated daily for 10 consecutive
days beginning 24 hours after tumor cell inoculation. Mean survival time was used
as an index of drug activity.
vitro cell systems
lung tumor: We obtained isolated Lewis lung tumor cells by subjecting 1-mm (third
power) sections of tumor to 0.25% trypsin at 22 degrees C and stirring for 60-90
minutes. After trypsinization, the cells were centrifuged (1,000 rpm for 10 min)
and washed twice in Dulbecco's medium containing 20% heat-inactivated fetal calf
serum. They were then reconstituted to 10 7 cells/ml of 200 mm glutamine, 5,000
U penicillin, and 5,000 mcg streptomycin. Tumor cells (3-6 ml) were dispensed
into 25-ml Erlenmeyer flasks and preincubated with eithe the drug or the drug
vehicle for 15 minutes in a Dubnoff metabolic shaker at 37 degrees C in an atmosphere
of 5% CO2--95% )2. After preincubation, 10 ucl tritiated thymidine (3H-TDR) (10
uCi, 57 Ci/mmole; New England Nuclear Corp., Boston, Mas.) was added to each flask
and incubated for various times, after which 1-ml aliquots were removed and placed
in 10 X 75-mm test tubes containing 1 ml 10% trichloroacetic acid (TCA) at 4 degrees
C. The TCA-precipated samples were then filtered on 0.45-u Millipore filters and
washed twice with 5 ml of 10% TCA at 4 degrees C. The filters were transferred
to liquid scintillation vials and counted in a toluene cocktail containing Liquifluor
(New England Nuclear Corp.) (4 liters toluene to 160 ml Liquifluor). Samples were
then counted in a liquid scintillator.
marrow: Bone marrow cells were derived from the tibias and fibulas of BDF 1 mice.
One ml Dulbecco's medium containing 1 U heparin/ml was forced through each bone
by a 1-ml syringe with a 26-gauge needle. The cells were washed three times, nucleated
cells were enumerated on a hemocytometer, and cell viability was ascertained by
trypan blue exclusion. Cell number was adjusted to 10 (seventh) cells/ml with
heparin-free Dulbecco's medium and incubated at 4 degrees C for 15 minutes. Bone
marrow cells were then dispensed (3-5 ml) into a25-ml Erlenmeyer flasks containing
the test drug or the drug vehicle. This preincubation period was followed by the
addition of 10 ul 3H-TDR and the procedures done as outlined for the isolated
Lewis lung cells.
L1210 cells were derived from DBA/2 mice as described above. They were obtained
from DBA/2 mice and inoculated 7 days before the experiment by the peritoneal
cavity being flushed with 10 ml Dulbecco's medium containing heparin (5 u/ml).
The cells were washed three times in medium, and the final medium wash did not
contain heparin. The cells were resuspended at 10 (seventh) cells/ml and treated
as described above. Cells were routinely counted with a hemocytometer for the
determination of cell viability with trypan blue; for Lewis lung tumor and L1210
cells, a Coulter apparatus (Mode ZB1) was also used. All other reagents were of
the highest quality grade available. Actinomycin D, 5-fluorouracil (5-FU), and
cytosine arabinoside (ara-C) were provided by the Drug Development Branch, National
Cancer Institute (NCI).
The structures of the four compounds are shown in text-figure 1.
All occur naturally in marihuana and were chemically synthesized. These drugs
were provided by the National Institute on Drug Abuse or the Sheehan Institute
for Research, Cambridge, Massachusetts. In the preparation of the drugs, the cannabinoids
were complexed to albumin or solubilized in Emulphor-alcohol. Both preparations
produced similar antitumor activity. With albumin, the cannabinoids were prepared
in the following manner: A stock solution of 150 mg cannabinoid per ml absolute
ethanol was made. Six ml of this solution was placed in a 200-ml flask. The ethanol
was evaporated off under a stream of nitrogen and 2,100 mg lyophilized bovine
serum albumin (BSA) added. After the addition of 20 ml distilled water, the substances
were stirred with a glass rod in a sonicator until a good suspension was achieved.
Sufficient distilled water was the aldded to make the desired dilution. Concentrations
were routinely checked with a gas chromatograph. When Emulphor-alcohol was used
as the vehicle, the desired amount of cannabinoid was sonicated in a solution
of equal volumes by absolute ethanol and Emulphor (El-620; GAF Corp., New York,
N.Y.) and then diluted with 0.15 N NaCL for a final ratio of 1: 1: 4 (ethanol:
of Cannabinoids on Murine Tumors
delta-8-THC, and cannabinol (CBN) all inhibited primary Lewis lung tumor growth,
whereas cannabidiol (CBD) enhanced tumor growth.
Oral administration of 25, 50, or 100 mg delta-9-THC/kg inhibited primary tumor
growth by 48, 72, and 75% respectively, when measured 12 days post tumor inoculation
On day 19, mice given delta-9-THC had a 34% reduction in primary tumor size. On
day 30, primary tumor size was 76% that of controls and only those given 100 mg
delta-9-THC/kg had a significant increase in survival time (36%). Mice treated
with a delta-9-THC showed a slight weight loss over the 2-week period (average
loss, 0.3 g at 50 mg/kg and 0.1 g at 100 mg/kg). This can be compared to cyclo-ohosphamide,
which caused weight loss approaching 20% (table 2).
activity was similar to that of delta-9-THC when administered orally daily until
death (table 2). However, as with delta-9-THC, primary tumor growth approached
control values after 3 weeks. When measured 12 days post tumor inoculation, all
doses (50-400 mg/kg) of delta-8-THC inhibited primary tumor growth between 40
and 60%. Significant inhibition was also seen on day 21, which was comparable
to cyclophosphamide-treated mice. Although this was not the optimum regime for
cyclophosphamide, it was the positive control protocol provided by the NCI (11).
All mice given delta-8-THC survived significantly longer than controls, except
those treated with 100 mg/kg. Mice given 50, 200, and 400 mg/kg delta-8-THC had
an increased life-span of 22.6, 24.6, and 27.2%, respectively, as compared to
33% for mice treated with 20 mg cyclophosphamide/kg. Pyran copolymer, an immunopotentiator
(12) when administered at 50 mg/kg, also significantly increased the survival
time of the animals (39.3%).
administered by gavage daily until death, demonstrated antitumor activity against
the Lewis lung carcinoma when evaluated on day 14 post tumor inoculation (table
Primary tumor growth was inhibited by 77%, at doses of 100 mg/kg on day 14 but
only by 11% on day 24. At 50 mg/kg on day 14 but only by 11% on day 24. At 50
mg/kg, CBN inhibited primary tumor growth by only 32% when measured on day 14,
and no inhibition was observed on day 24; however, these animals did survive 27%
CBD, administered at 25 or 200 mg/kg daily until death, showed no tumor-inhibitory
properties as measured by primary Lewis lung tumor size or survival time (table
In this experiment, CBD-treated mice showed enhanced primary tumor growth. However,
the control tumor growth rate in this experiment was decreased as compared to
the previous studies.
time of BDF 1 mice hosting L1210 leukemia was not prolonged by delta-9-THC treatment
Mice treated with delta-9-THC at doses of 50, 100, and 200 mg/kg administered
orally daily until death, survived 8.5, 7.8, and 8.6 days, respectively, as compared
to 8.6 days for mice treated with the diluent. However, delta-9-THC inhigited
FLV-induced splenomegaly by 71% at 200 mg/kg as compared to 90.2% for the positive
control actinomycin D (0.25 mg/kg). Although there was a dose-related inhibition,
only the high dose was statistically significant (table 6).
of Cannabinoids on Isolated Cells In Vitro
cells incubated in vitro represent a simple, reliable, and, hopefully, predictive
method for the monitoring of the effects of agents on several biochemical parameters
at the same time. The incorporation of 3H-TDR into TCA-precipitable counts in
isolated Lewis lung cells is shown in text-figure 2. Similar types of curves were
seen for bone marrow and L1210 cells. In all instances, for 15-45 minutes there
was a linear increase in 3H-TDR uptake into the TCA-precipitable fraction. Qualitatively,
similar data (not shown) were seen after a pulse with 14C-uridine. Actinomycin
D (1 mcg/ml) preferentially inhibited 14C-uridine incorporation after uridine
uptake had decreased to less than 30% that of control (data not shown). This is
indirect evidence that we were measuring RNA synthesis.
Experiments (data not shown) done with 5-FU (10 -4 M) indicated that, in isolated
bone marrow cells, both thymidine uptake with time by delta-9-THC (10 -5 M) on
Lewis lung cells is depicted in text-figure 2.
this experiment, delta-9-THC caused a nonlinear uptake of 3H-TDR. At 30 minutes,
uptake of 3H-TDR into the acid-precipitable fraction was about 50% that of control
Longer incubations (i.e., 60 min) did not significantly change the uptake pattern
for control and de;ta-9-THC treated tumor cells. The effect of several cannabinoids
on the uptake of 3H-TDR into cells incubated in vitro indicated that delta-9-THC,
delta-8-THC, and CBN produced a dose-dependent inhibition of radiolabel uptake
in the three cell types (table 7). These results, presented as percent inhibition
of radiolabel uptake as compared to control, represented an effect of cannabinoids
on one aspect of macromolecular synthesis.
was the least active of the cannabinoids, but showed its greatest activity in
the L1210 leukemia cells. Other data (not shown) indicate that these compounds
similarly effect the uptake of 14C-uridine into the acid-precipitable fraction.
Ara-C markedly inhibited 3H-TDR uptake more dramatically than did the cannabinoids
(table 7). Note that delta-9-THC exhibited inhibitory properties in the isolated
Lewis lung tumor and L1210 cells at concentrations that did not interfere with
thymidine uptake into bone marrow cells. At certain concentrations of CBD (2,5
X 10 -6 and 2.5 X 10 -7M), radiolabel uptake was consistently stimulated in bone
marrow cells and in several experiments with the isolated Lewis lung cells.
investigated four cannabinoids for antineoplastic activity against three animal
tumor models in vivo and for cytotoxic or cystostatic activity in two tumor cell
lines and bone marrow cells in vitro.
cannabinoids (delta-9-THC, delta-8-THC, and CBN) active in vivo against the Lewis
lung tumor cells are also active in the in vitro systems. The differential sensitivity
of delta-9-THC against Lewis lung cells versus bone marrow cells is unique in
that delta-8-THC and CBN are equally active in these systems. Johnson and Wiersma
(5) reported that delta-9-THC administered iv caused a reduction in bone marrow
metamyelocytes and an increase in lymphocytes. It is unclear from the data whether
this is a depression of myelopoiesis or if it represents a lymphocyte infiltration
into the bone marrow. The use of isolated bone marrow cells, which represent a
nonneoplastic rapidly proliferating tissue, enables the rapid evaluation and assessment
of drug sensititity and specificity, and thereby may predict toxicity related
to bone marrow suppression.
showed noninhibitory activity either against the Lewis lung cells in vivo or Lewis
lung and bone marrow cells in vitro at 10 -5M an 10 -6M, respectively. Indeed,
the tumor growth rate in mice treated with CBD was significantly increased over
controls. This may, in part, be the consequence of the observation made in vitro
(i.e., 10 -7M CBD stimulated thymidine uptake), which may be reflected by an increased
rate of tumor growth. One problem related to the use of cannabinoids is the development
of tolerance to many of its behavioral effects (13). It also appears that tolerance
functions in the chemotherapy of neoplsms in that the growth of the Lewis lung
tumor is initially markedly inhibited but, by 3 weeks, approaches that of vehicle-treated
mice (tables 1, 3). This, in part, may reflect drug regimens, doses used, increased
drug metabolism, or conversion to metabolites with antagonistic actions to delta-9-THC.
It may also represent some tumor cell modifications rendering the cell insensitive
to these drugs.
further interest was the lack of activity of delta-9-THC against the L1210 in
vivo, whereas the invitro L1210 studies indicated that delta-9-THC could effectively
inhibit thymidine uptake. The apparent reason for this discrepancy may be related
to the high growth fraction and the short doubling time of this tumor. The in
vitro data does not indicate that the cannabinids possess that degree of activity;
e.g., ara-C, which "cures" L1210 mice, is several orders of magnitude
more potent on a molar basis than delta-9-THC in vitro.
of tumor growth and increased animal survival after treatment with delta-9-THC
may, in part, be due to the ability of the drug to inhibit nucleic acid synthesis.
Preliminary data with Lewis lung cells grown in tissue culture indicate that 10
-5M delta-9-THC inhibits by 50% the uptake of 3H-TDR into acid-precipitable counts
over a 4-hour incubation period. Simultaneous determination of acid-soluble fractions
did not show any inhibitory effects on radiolabeled uptake. Therefore, delta-9-THC
may be acting at site(s) distal to the uptake of precursor. We are currently evaluating
the acid-soluble pool to see if phosphorylation of precursor is involved in the
action of delta-9-THC.
results lend further support to increasing evidence that, in addition to the well-known
behavioral effects of delta-9-THC, this agent modifies other cell responses that
may have greater biologic significance in that they have antineoplastic activity.
The high doses of delta-9-THC (i.e., 200 mg/kg) are not tolerable in humans. On
a body-surface basis, this would be about 17 mg/m(2) for mice. Extrapolation to
a 60-kg man would require 1,020 mg for comparable dosage. The highest doses administered
to man have been 250-300 mg (14). Whether only cannabinoids active in the central
nervous system (CNS) exhibit this antineoplastic property is not the question,
since CBN, which lacks marihuana-like psychoactivity, is quite active in our systems
structure-activity investigations, more active agents may be designed and synthesized
which are devoid of or have reduced CNS activity. That these compounds readily
cross the blood-brain barrier and do not possess many of the toxic manifestations
of presently used cytotoxic agents, makes them an appealing group of drugs to
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