Cannbinoids reduce tumors in mice


ANTINEOPLASTIC ACTIVITY OF CANNABINOIDS

A.E. Munson, L.S. Harris, M.A. Friedman, W.L. Dewey, and R.A. Carchman*

Journal of the National Cancer Institute, Vol. 55, No. 3, September 1975

Supported 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 Cancer Society.

Department of Pharmacology and the MCV/VCU Cancer Center, Medical College
of Virginia, Virginia Commonwealth University. Richmond, Va. 23298

Summary----Lewis 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). ----J Natl Cancer Inst 55: 597-602, 1975.

Investigations 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 and spermatogenesis. 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 of action(s).

MATERIALS AND METHODS

The tumor systems used were the Lewis lung adenocarcinoma, leukemia
L1210, and B-tropic Friend leukemia.
In vivo systems.---Lewis 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).
Friend 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.
L1210 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.
In vitro cell systems. ---Lewis 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.
Bone 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: 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).
Cannabinoids. ---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: Emulphor: NaCL).

RESULTS

Effects of Cannabinoids on Murine Tumors

Delta-9-THC, 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 (table 1). 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).
Delta-8-THC 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 regim;en
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%).
CBN, administered by gavage daily until death, demonstrated
antitumor activity against the Lewis lung carcinoma when evaluated on day
14 post tumor inoculation (table 3). 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% longer.
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 4). 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.
Survival time of BDF 1 mice hosting L1210 leukemia was not
prolonged by delta-9-THC treatment (table 5). 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).

Effect of Cannabinoids on Isolated Cells In Vitro

Isolated 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. In 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 effectof
cannabinoids on one aspect of macromolecular synthesis. CBD 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.

DISCUSSION

We 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. The
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. CBD 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. Of 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 do 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.
Inhibition 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.
These 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 (15). With
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 study.

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