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ORIGINAL ARTICLE
Eduardo Lasalvia-Prisco Æ Silvia Cucchi
Jesu
´
sVa
´
zquez Æ Eduardo Lasalvia-Galante
Wilson Golomar Æ William Gordon
Insulin-induced enhancement of antitumoral response
to methotrexate in breast cancer patients
Received: 31 March 2003 / Accepted: 29 August 2003 / Published online: 4 December 2003
Springer-Verlag 2003
Abstract Purpose: It has been reported that insulin in-
creases the cytotoxic effect in vitro of methotrexate by as
much as 10,000-fold. The purpose of this study was to
explore the clinical value of insulin as a potentiator of
methotrexate. Patients and methods: Included in this
prospective, randomized clinical trial were 30 women
with metastatic breast cancer resistant to fluorouracil +
Adriamycin + cyclophosphamide and also resistant to
hormone therapy with measurable lesions. Three groups
each of ten patients received two 21-day courses of the
following treatments: insulin + methotrexate, metho-
trexate, and insulin, respec tively. In each patient, the size
of the target tumor was measured before and after
treatment according to the Response Evaluation Criteria
In Solid Tumors. The changes in the size of the target
tumor in the three groups were compared statistically.
Results: Under the trial conditions, the methotrexate-
treated group and the insulin-treated group responded
most frequently with progressive disease. The group
treated with insulin + methotrexate responded most
frequently with stable disease. The median increase in
tumor size was significantly lower with insulin +
methotrexate than with each drug used separately.
Discussion: Our results confirmed in vivo the results of
previous in vitro studies showing clinical evidence that
insulin potentiates methotrexate under conditions where
insulin alone does not promote an increase in tumor
growth. Therefore, the chemotherapy antitumoral
activity must have been enhanced by the biochemical
events elicited in tumor cells by ins ulin. Conclusions:In
multidrug-resistant metastatic breast cancer, metho-
trexate + insulin produced a significant antitumoral
response that was not seen with either methotrexate or
insulin used separately.
Keywords Breast Cancer Æ Chemotherapy Æ Insulin Æ
Methotrexate Æ Tumor growth
Introduction
It is known that slowly growing cancers have tumor cell
populations with a low-growth fraction and are less
sensitive to chemotherapy than rapidly growing tumors
with high-growth fractions [11]. Slowly growing malig-
nancies have relatively more cells in a noncycling status
and fewer cells in a cycling status than rapidly growing
malignancies. It has been demonstrated that insulin as a
pharmacological agent induces the switch from a non-
cycling to a cycling status in tumor cells [5]. In MCF-7
human breast cancer cells, insulin has been shown to
increase the cytotoxic effect of methotrexate up to
10,000-fold in vitro [1]. Ellipticine uptake is also
increased by ins ulin [9]. It has been suggested that
insulin is effective in potentiating most chemotherapy
drugs. This insulin-induced potentiation has been
Cancer Chemother Pharmacol (2004) 53: 220–224
DOI 10.1007/s00280-003-0716-7
E. Lasalvia-Prisco
Department of Medicine, School of Medicine,
University of Uruguay, Montevideo, Uruguay
S. Cucchi Æ J. Va
´
zquez Æ E. Lasalvia-Galante Æ W. Golomar
E. Lasalvia-Prisco
Interdoctors Medical Center,
Montevideo, Uruguay
E. Lasalvia-Prisco Æ J. Va
´
zquez Æ E. Lasalvia-Galante
W. Golomar
National Cancer Institute,
Montevideo, Uruguay
S. Cucchi Æ W. Gordon
PharmaBlood Inc,
North Miami Beach, Florida, USA
E. Lasalvia-Prisco (&)
(Former Director of the Department of Medicine,
School of Medicine, University of Uruguay,
Montevideo, Uruguay and National Cancer Institute,
Montevideo, Uruguay),
Research & Development Department,
PharmaBlood Inc, 2050 NE 163rd Street,
2nd Fl, 202, North Miami Beach,
Florida, 33162, USA
E-mail: research@pharmablood.com
Tel.: +1-305-9442544
Fax: +1-305-9445244
proposed as a strategy for breast cancer treatment, but
confirmatory clinical trials are still lacking [2]. This
study was carried out to confirm insulin-induced clinical
potentiation of the antitumoral effect of methotrexate as
suggested by preclinical studies and to establish a
mechanism of action for this antitumoral effect.
Patients and methods
Patients
The study was conducted in 30 patients with breast cancer admitted
to medical centers that reported medical data to the Cooperative
Trials Center (CTC) of PharmaBlood, R&D Department, Florida.
A prospective, randomized trial was carried out. All patients met
the following eligibility criteria: histologically confirmed breast
carcinoma, metastatic stage (M1); Eastern Cooperative Oncology
Group (ECOG) performance status (PS) £ 2; age £ 74 years; and
adequate hematological function (WBC count ‡4000/ll, neutrophil
count ‡2000/ll, hemoglobin level ‡9.0 g/dl, platelet count
‡10·10
4
/ll), renal function (serum creatinine £ 1.5 mg/dl, 24-h
creatinine clearance £ 60 ml/min), liver function (total bilirubin
£ 2.0 mg/dl, serum transaminases not more than twice the upper
limit of the normal range), and respiratory function (PaO
2
‡60 Torr). The patients included had measurable lesions, as
required by the Response Evaluation Criteria In Solid Tumors
(RECIST) system of tumor assessment [13], and if they had a
positive estrogen receptor status, they had been treated with and
become resistant to hormone therapy.
All patients included in the study had progressive disease
(RECIST criteria) after chemotherapy with at least four series of
fluorouracil + Adriamycin + cyclophosphamide (FAC) and had
not been treated with any other chemotherapy. They were ran-
domly allocated to three groups of ten patients each: group 1 was
treated with insulin + methotrexate as described below, group 2
was treated with methotrexate without insulin, and group 3 was
treated with insulin without methotrexate. Written informed con-
sent, including detailed information about risks and benefits, was
approved and signed by all the patients included in the study.
Central computerized remote randomization was performed, with
patients being allocated to one of the groups through random se-
quence generation by the permuted block method. An assessment
of the results after 30 patients had completed the trial showed that
this sample size was enough. The patients were recruited from two
oncological medical centers in Montevideo, Uruguay (first at the
National Cancer Institute and then at Interdoctors Medical Cen-
ter), both of which participated with their data in the network
operated and sponsored by the Cooperative Trials Center (CTC) of
PharmaBlood R&D Department.
The institutional ethics committee of PharmaBlood and the
institutional review boards of the participating medical centers
approved the trial. The ethical reviewers considered that an 8-week
delay before starting second-line chemotherapy after FAC had
failed in all the patients included in the trial was acceptable. This
determination was consistent with the standard of care in this
clinical situation which has been recently well summarized [3]:
Despite almost 30 years of clinical cancer research, the true
impact of second and subsequent lines of chemotherapy on the
outcome of metastatic breast cancer patients, especially on the
duration of survival, is still unknown. In the virtually incurable
metastatic setting, issues like quality of life and patients pref-
erences gain particular relevance.
The accepted protocol was resubmitted to the committee for
review in order to obtain approval for treatment of patients with
insulin alone considering the potentially harmful effect through the
activation of receptors for insulin/insulin-like growth factors. The
committee confirmed the approval on the basis of reports of no
harmful effect of this treatment [6, 7]. The results of the study
confirmed the committees criteria because no significant differences
were found in tumor growth either between the insulin-alone group
and the methotrexate-alone group or between before and after
treatment in the insulin-alone group.
Treatment
All the patients included in the study received two 21-day courses
of treatment separated by a 7-day interval without treatment be-
tween courses. In group 1, the treatment course was intravenous
human recombinant insulin (0.3 U/kg body weight every other
day) followed 20 min later by a 15-min intravenous infusion of
methotrexate (2.5 mg/m
2
in 50 ml 30% glucose). If symptomatic
hypoglycemia was observed, the 30% glucose solution containing
methotrexate was infused immediately. An oral glucose supplement
was also prescribed to prevent delayed hypoglycemic symptoms. In
group 2, insulin was omitted and methotrexate was administered
intravenously at the same dose and in the same solution (2.5 mg/m
2
in 50 ml 30% glucose) as in group 1. In group 3, methotrexate was
omitted, insulin was administered at the same dose as in group 1,
and 30% glucose solution was also administered intravenously
20 min after insulin or sooner if hypoglycemic symptoms were
evident.
Tumor growth assessment
After 8 weeks (two 3-week courses plus 1 week interval after each
course), the response to treatment was assessed in each patient
using RECIST criteria [13]. The sum of the longest diameter of
measurable target lesions and the number of non-target lesions
were recorded immediately before and after this 8-week period.
Skin nodules and palpable lymph nodes were measured using
calipers. Lung and liver target lesions were measured by a CAT
scan. Responses were confirmed by repeating the assessment
4 weeks after status assignment. Three independent reviewers per-
formed all image measures (Telemedical Organization, North
Miami Beach, Fl.).
The distribution of RECIST status (progressive disease, stable
disease, or remission) in each group was recorded. This distribution
was dependent on treatments that showed statistical significance
according to the Chi-squared test. The data from the RECIST
measurements of the change in tumor size of the patients in each
treatment group, expressed as a percentage of pretreatment
measurements, were compared using Students t-test. Additionally,
increases in tumor size were expressed as a proportion of the initial
value and analyzed by the two-proportion test comparing pairs of
groups: group 3 vs group 1, and group 2 vs group 1. The sample
size was assessed after analysis of the results when the trial was
finished for the 30 patients allocated to the three groups. The above
pairs of groups were analyzed for the proportion of progressive
disease in each. Ten patients in each group was the required sample
size for an 80% chance of rejecting the hypothesis of equal pro-
portions at the 0.05 level of significance when the true proportions
were those shown by the study. Statistical analysis was performed
using StatsDirect software and an independent expert was con-
sulted.
Results
The characteristics of the patients included are shown in
Table 1. The three groups were comparable in the most
relevant prognostic parameters for the clinical condition
studied. Previous treatments were also comparable. The
similar range of sizes of target lesions measured before
treatment was especially significant, allowing the change
in size to be measured as a percentage of initial size.
221
Figure 1 shows the RECIST status assessed und er the
study conditions. Progressive disease was the most fre-
quent response in two of the three groups: in group 2
(treated with methotrexate alone) there were seven
progressive disease and three stable disease, and in
group 3 (treated with insulin alone) there were eight
progressive disease and two stable disease. In group 1
(treated with insulin + methotrexate), stable disease was
the most frequent response (nine stable disease, one
progressive disease). The distribution of RECIST type
responses (stable disease and progressive disease) was
dependent on the treatments tested, and was statistically
significant (P<0.01, Chi-squared test).
Figure 2 shows the means and 95% confidence
intervals (CI) of the percentage increase in tumor size
after treatment in the three groups. Incre ases in tumor
size were significantly lower in patients treated with
insulin + methotrexate than in those treated with
insulin alone and significantly lower than in those trea-
ted with methotrexate alone.
From the same set of measurements, Figs. 1 and 2
show the clinical and biological effects of the treatments,
respectively. Figure 1 indicates that the decrease in tu-
mor growth induced by insulin + methotrexate reached
the level of a clinically confirmed antitumoral response
because more patients in this group achieved stable
disease. Figure 2 shows that insulin + methotrexate
treatment reduced tumor growth. All patients completed
the study. Hypoglycemia was induced in all patients
receiving insulin as part of their protocol. Eight patients
in group 1 and nine patients in group 3 showed no
hypoglycemic symptoms during the 20 min after insulin
injection; they showed a mean blood glucose level of
456 mg/dl (range 376–520 mg/dl). Two patients in group
Table 1 Clinical characteristics
of the 30 women with
metastatic breast cancer (M1)
included in the three treatment
groups
Group 1
(insulin + methotrexate)
Group 2
(methotrexate)
Group 3
(insulin)
No. of patients 10 10 10
Age range (years) 42–64 44–68 39–69
<50 years 4 3 4
Estrogen receptor-positive 7 7 6
Progesterone receptor-positive 7 5 7
Measurable M1
Lung 6 4 4
Liver 1 2 2
Skin 2 2 3
Lymph nodes 1 2 1
Range of initial (pretreatment)
RECIST sum of target
measures (mm)
57–65 59–64 56–66
Fig. 1 Post-treatment RECIST status of measurable target lesions.
After the respective treatment, the change in the measurable lesions
selected as targets in each patient was evaluated and the status of
therapeutic response, defined in terms of the RECIST criteria, was
recorded. Under the conditions of this study, two response statuses
were recorded: stable disease (less than 20% increase or less than
30% decrease in the sum of largest diameters of targets) and
progressive disease (more than 20% increase in the sum of
diameters). Stable disease, the best response obtained, was more
frequent in the group treated with insulin + methotrexate (nine of
ten) than in methotrexate-treated group (three of ten) or insulin-
treated group (two of ten). The distribution of RECIST type
responses (stable disease or progressive disease) was dependent on
the treatments tested and statistically significant (P<0.01, Chi-
squared test)
Fig. 2 Increase in size of measurable target lesions (RECIST
assessment). After each treatment, the change in the measurable
lesions selected as targets in each patient was evaluated in terms of
the RECIST criteria and expressed as a percentage of the measured
pretreatment size. For each treatment group, the mean±SD and
95% CI for the values of this response were calculated: group 1
(insulin + methotrexate) 13.51±3.01% (95% CI 11.35–15.67%);
group 2 (methotrexate) 20.21±2.27% (95% CI 18.58–21.84%);
group 3 (insulin) 21.04±2.17% (95% CI 19.49–22.59%). The
increase in size of lesions in group 1 (insulin + methotrexate) was
significantly lower (Students t-test) than the increase in size in
group 2 (methotrexate) (P<0.001) and group 3 (insulin)
(P<0.001). Group 2 showed no significant difference from group
3(P=0.41)
222
1 and one patient in group 3 showed hypoglycemic
symptoms within 20 min of insulin injection (13, 16 and
19 min), but recovered immediately after starting the
glucose infusion. There was no evidence of any harmful
sequelae attributable to the hypoglycemia induced.
Table 2 shows the toxicities associated with antitu-
moral chemotherapy (according to WHO criteria)
recorded in this study.
Discussion
The methotrexate dose used in this study was chosen
because a similar dose of methotrexate had been used
previously in patients receiving low-dose combined
chemotherapy potentiated with insulin [2]. In addition,
the cumulative monthly dose was no higher than the
monthly dose used in the well-known standard protocol
of methotrexate + fluorouracil + cyclophosphamide
(CMF). Indeed, each individual methotrexate injection
(2.5 mg/m
2
) was less than the dose usually co nsidered
optimal in non-potentiated protocols but is within the
presumed range of effective dose for a potentiation
similar to the one observed in vitro. The results of this
study confirmed the expected safety of the selected
methotrexate dose. The toxicities in the methotrexate-
alone group were not relevant (WHO grades 1/2) and
they were even lower when methotrexate was associated
with insulin, only producing a grade 1 mucositis. In this
study, methotrexate at this safe low dose did not have an
antitumoral effect when used alone (group 2), but it did
produce a significant antitumoral effect when adminis-
tered after insulin (group 1). The term antitumoral is
used here as a description of the clinical effect of a
reduction in the proportion of patients showing pro-
gressive disease.
Therefore, as rep orted previously, our results support
the hypothesis that insulin can potentiate the antitu-
moral effect of methotrexate [2] and confirm in vivo
previously reported in vitro results [10]. Our results also
show insulin potentiation of methotrexate in this con-
dition, where insulin alone did not promote an increase
in tumor growth (group 3). This effect is in agreement
with previous results from in vitro models where insulin
enhancement of cytotoxicity was not a direct conse-
quence of an insulin-dependent increase in the growth
rate of tumor cells [1, 10]. The same in vitro models do
not allow an explanation of the insulin potentiation of
methotrexate in terms of the known effects of insulin
treatment upon the specific metab olism of methotrexate
which include a decrease in intracellular pH induced by
glucose metabolism and tight binding of the drug to its
target, dihydrofolate reductase. Insulin pote ntiation of
other antitumoral drugs has been reported [9].
If we discount the promotion of tumor cell growth
and the interaction with the specific target as the
mechanism of potentiation of methotrexate by insulin,
we can hypothesize that this mechanism could involve
another general insulin-dependent biochemical pathway
as has been previously suggested to explain the in vitro
potentiation of methotrexate by insulin [1]: protein
synthesis in tumor cells is one of the biochemical path-
ways activated by insulin [8]. Most chemotherapy drugs
that have been tested using insulin to increase cytotox-
icity are known modifiers of protein stru cture that act at
the genetic or epigenetic level [12]. High levels of mu-
tated or epigenetically modified proteins could be
responsible for the cytotoxic mechanism elicited by the
insulin-dependent increase in protein synthesis associ-
ated with chemotherapy drugs. The relative selectivity of
this mechanism of action for insulin + methotrexate in
malignant cells is attributed to the agonism of insulin
and insulin-like receptors in tumor cells. Certainly, the
response to insulin is more intense in most tested cancer
cells than in most normal cells. This is probably because
cancer cells are richer in receptors for insulin-like growth
factors that are cross-stimulated by insulin [4].
Conclusion
The in vitro potentiation of methotrexate cytotoxicity by
insulin in human breast cancer cell lines was previously
known. We report the results of a randomized, co n-
trolled trial that confirmed, at the clinical level, the
potentiation by insulin of the antitumoral effect of
methotrexate in women with advanced breast cancer.
The term antitumoral is used as a description of the
clinical effect of a reduction in the proportion of patients
with progressive disease. Under the conditions of this
study, the dose of insulin used did not increase tumor
growth. Therefore, we suggest that, as has been reported
Table 2 Maximum recorded WHO toxicity grade in the patients
included in the trial comparing insulin + methotrexate (group 1),
methotrexate (group 2) and insulin (group 3). The numbers of
patients with each toxicity grade (0 to 4) in the three groups are
shown. No other toxicities referred to in the WHO criteria were
recorded
Toxicity Grade
01234
Erythrocytes
Group 1 10 0 0 0 0
Group 2 8 2 0 0 0
Group 3 10 0 0 0 0
Leukocytes
Group 1 10 0 0 0 0
Group 2 6 3 1 0 0
Group 3 10 0 0 0 0
Platelets
Group 1 10 0 0 0 0
Group 2 9 1 0 0 0
Group 3 10 0 0 0 0
Mucositis
Group 1 8 2 0 0 0
Group 2 4 3 3 0 0
Group 3 10 0 0 0 0
223
in vitro, methotrexate potentiation by insulin was not a
direct consequence of the expansion of the tumor cycling
cell population but a consequence of some of the bio-
chemical events that are simultaneously activated. The
enhancement of methotrexate uptake by tumor cells
and/or the promotion of protein synthesis in a muta-
genic intracellular environme nt are hypothesized to be
mechanisms of potentiation. It is known that both
events are promoted by insulin acting as a cross-agonist
of the highly expressed receptors for insulin-like growth
factors in breast cancer cells.
These mechanisms, which are shared with other pri-
mary tumor cells and with other chemotherapeutic
agents suggest that it would be worthwhile to pursue
further study of these phenomena in other tumors and
with other chemotherapeutic agents.
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