Alterations of antitumor and metabolic responses in L5178Y-R lymphoma-bearing mice after only 30-minute daily chronic stress exposure
Aim: In stress research, reducing times of stress induction may contribute to improving the well-being of experimental animals, especially in cancer models, already under physiological distress. To support this idea, we evaluated the effects of a short-timed stress protocol on endocrine, metabolic a...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
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| Цитувати: | Alterations of antitumor and metabolic responses in L5178Y-R lymphoma-bearing mice after only 30-minute daily chronic stress exposure / D. Caballero-Hernandez, D. Najera-Valderrabano, A. Valadez-Lira, M. Franco-Molina, R. Gomez-Flores, P. Tamez-Guerra, R. Tamez-Guerra, C. Rodríguez-Padilla // Experimental Oncology. — 2017 — Т. 39, № 4. — С. 276–280. — Бібліогр.: 27 назв. — англ. |
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irk-123456789-1385882018-06-20T03:06:08Z Alterations of antitumor and metabolic responses in L5178Y-R lymphoma-bearing mice after only 30-minute daily chronic stress exposure Caballero-Hernandez, D. Najera-Valderrabano, D. Valadez-Lira, A. Franco-Molina, M. Gomez-Flores, R. Tamez-Guerra, P. Tamez-Guerra, R. Rodríguez-Padilla, C. Original contributions Aim: In stress research, reducing times of stress induction may contribute to improving the well-being of experimental animals, especially in cancer models, already under physiological distress. To support this idea, we evaluated the effects of a short-timed stress protocol on endocrine, metabolic and immune indicators in mice bearing the L5178Y-R lymphoma. Materials and Methods: A 30-minute daily stress protocol was applied for 28 days to healthy and lymphoma-bearing BALB/c mice; body weight, plasma levels of corticosterone, norepinephrine, Th1/Th2 cytokines, insulin, and leptin, were measured. Results: We found a 12% significant decrease in body weight in non-tumor bearing mice under stress (p < 0.007). The disruption of weight evolution was accompanied by a stress induced 85% decrease in plasmatic leptin (p < 0.01) and total reduction of insulin. Tumor burden alone was associated to an increase in more than two-fold of plasmatic levels of norepinephrine (p < 0.008). Neither stress nor tumor or their combination, resulted in an elevation of systemic IL-6. IFN-γ levels were 20 times higher in lymphoma-bearing animals when compared with non-tumor bearing mice (p < 0.01); however, under stress, this response was reduced by half, indicating a suppressing effect of chronic stress on the antitumor immune response. Conclusion: A short-timed stress induction is enough to cause significant alterations in the metabolism and immunity of healthy and tumor-bearing mice, supporting the use of short-timed protocols as an efficient way to induce chronic stress that also considers concerns regarding the well-being of experimental animals in biomedical research. 2017 Article Alterations of antitumor and metabolic responses in L5178Y-R lymphoma-bearing mice after only 30-minute daily chronic stress exposure / D. Caballero-Hernandez, D. Najera-Valderrabano, A. Valadez-Lira, M. Franco-Molina, R. Gomez-Flores, P. Tamez-Guerra, R. Tamez-Guerra, C. Rodríguez-Padilla // Experimental Oncology. — 2017 — Т. 39, № 4. — С. 276–280. — Бібліогр.: 27 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/138588 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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English |
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Original contributions Original contributions |
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Original contributions Original contributions Caballero-Hernandez, D. Najera-Valderrabano, D. Valadez-Lira, A. Franco-Molina, M. Gomez-Flores, R. Tamez-Guerra, P. Tamez-Guerra, R. Rodríguez-Padilla, C. Alterations of antitumor and metabolic responses in L5178Y-R lymphoma-bearing mice after only 30-minute daily chronic stress exposure Experimental Oncology |
| description |
Aim: In stress research, reducing times of stress induction may contribute to improving the well-being of experimental animals, especially in cancer models, already under physiological distress. To support this idea, we evaluated the effects of a short-timed stress protocol on endocrine, metabolic and immune indicators in mice bearing the L5178Y-R lymphoma. Materials and Methods: A 30-minute daily stress protocol was applied for 28 days to healthy and lymphoma-bearing BALB/c mice; body weight, plasma levels of corticosterone, norepinephrine, Th1/Th2 cytokines, insulin, and leptin, were measured. Results: We found a 12% significant decrease in body weight in non-tumor bearing mice under stress (p < 0.007). The disruption of weight evolution was accompanied by a stress induced 85% decrease in plasmatic leptin (p < 0.01) and total reduction of insulin. Tumor burden alone was associated to an increase in more than two-fold of plasmatic levels of norepinephrine (p < 0.008). Neither stress nor tumor or their combination, resulted in an elevation of systemic IL-6. IFN-γ levels were 20 times higher in lymphoma-bearing animals when compared with non-tumor bearing mice (p < 0.01); however, under stress, this response was reduced by half, indicating a suppressing effect of chronic stress on the antitumor immune response. Conclusion: A short-timed stress induction is enough to cause significant alterations in the metabolism and immunity of healthy and tumor-bearing mice, supporting the use of short-timed protocols as an efficient way to induce chronic stress that also considers concerns regarding the well-being of experimental animals in biomedical research. |
| format |
Article |
| author |
Caballero-Hernandez, D. Najera-Valderrabano, D. Valadez-Lira, A. Franco-Molina, M. Gomez-Flores, R. Tamez-Guerra, P. Tamez-Guerra, R. Rodríguez-Padilla, C. |
| author_facet |
Caballero-Hernandez, D. Najera-Valderrabano, D. Valadez-Lira, A. Franco-Molina, M. Gomez-Flores, R. Tamez-Guerra, P. Tamez-Guerra, R. Rodríguez-Padilla, C. |
| author_sort |
Caballero-Hernandez, D. |
| title |
Alterations of antitumor and metabolic responses in L5178Y-R lymphoma-bearing mice after only 30-minute daily chronic stress exposure |
| title_short |
Alterations of antitumor and metabolic responses in L5178Y-R lymphoma-bearing mice after only 30-minute daily chronic stress exposure |
| title_full |
Alterations of antitumor and metabolic responses in L5178Y-R lymphoma-bearing mice after only 30-minute daily chronic stress exposure |
| title_fullStr |
Alterations of antitumor and metabolic responses in L5178Y-R lymphoma-bearing mice after only 30-minute daily chronic stress exposure |
| title_full_unstemmed |
Alterations of antitumor and metabolic responses in L5178Y-R lymphoma-bearing mice after only 30-minute daily chronic stress exposure |
| title_sort |
alterations of antitumor and metabolic responses in l5178y-r lymphoma-bearing mice after only 30-minute daily chronic stress exposure |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2017 |
| topic_facet |
Original contributions |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/138588 |
| citation_txt |
Alterations of antitumor and metabolic responses in L5178Y-R lymphoma-bearing mice after only 30-minute daily chronic stress exposure / D. Caballero-Hernandez, D. Najera-Valderrabano, A. Valadez-Lira, M. Franco-Molina, R. Gomez-Flores, P. Tamez-Guerra, R. Tamez-Guerra, C. Rodríguez-Padilla // Experimental Oncology. — 2017 — Т. 39, № 4. — С. 276–280. — Бібліогр.: 27 назв. — англ. |
| series |
Experimental Oncology |
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2025-07-10T06:07:30Z |
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| fulltext |
276 Experimental Oncology 39, 276–280, 2017 (December)
ALTERATIONS OF ANTITUMOR AND METABOLIC RESPONSES
IN L5178Y-R LYMPHOMA-BEARING MICE
AFTER ONLY 30-MINUTE DAILY CHRONIC STRESS EXPOSURE
D. Caballero-Hernandez*, D. Najera-Valderrabano, A. Valadez-Lira, M. Franco-Molina,
R. Gomez-Flores, P. Tamez-Guerra, R. Tamez-Guerra, C. Rodríguez-Padilla
Department of Microbiology and Immunology, Laboratory of Immunology and Virology,
Faculty of Biological Sciences, Autonomous University of Nuevo León,
San Nicolás de los Garza, NL 66455, Mexico
Aim: In stress research, reducing times of stress induction may contribute to improving the well-being of experimental animals,
especially in cancer models, already under physiological distress. To support this idea, we evaluated the effects of a short-timed
stress protocol on endocrine, metabolic and immune indicators in mice bearing the L5178Y-R lymphoma. Materials and Methods:
A 30-minute daily stress protocol was applied for 28 days to healthy and lymphoma-bearing BALB/c mice; body weight, plasma
levels of corticosterone, norepinephrine, Th1/Th2 cytokines, insulin, and leptin, were measured. Results: We found a 12% significant
decrease in body weight in non-tumor bearing mice under stress (p < 0.007). The disruption of weight evolution was accompanied
by a stress induced 85% decrease in plasmatic leptin (p < 0.01) and total reduction of insulin. Tumor burden alone was associated
to an increase in more than two-fold of plasmatic levels of norepinephrine (p < 0.008). Neither stress nor tumor or their combina-
tion, resulted in an elevation of systemic IL-6. IFN-γ levels were 20 times higher in lymphoma-bearing animals when compared
with non-tumor bearing mice (p < 0.01); however, under stress, this response was reduced by half, indicating a suppressing effect
of chronic stress on the antitumor immune response. Conclusion: A short-timed stress induction is enough to cause significant altera-
tions in the metabolism and immunity of healthy and tumor-bearing mice, supporting the use of short-timed protocols as an efficient
way to induce chronic stress that also considers concerns regarding the well-being of experimental animals in biomedical research.
Key Words: psychogenic stress, tumor models, immunosuppression, glucose homeostasis, IFN-γ, leptin, animal welfare.
Psychogenic stress has been proven to have a signifi-
cant role in cancer initiation and progression; hence, the
study of its many biological effects is of great inte rest [1,
2]. Besides providing a rational basis for preventive
stress management, such studies may identify targets
for therapeutic intervention aiming at improving quality
of life in cancer patients. In this sense, an often used
pre-clinical approach to study the influence of stress
on cancer involves the application of a stress-inducing
paradigm in tumor-bearing animals [3]. One of the most
frequently used is the restraint stress paradigm, which
involves placing mice or rats in a small, closed container
to reduce its ability to move [4, 5]. Other paradigms,
such as forced swim and social isolation are also valuable
to dissect the mechanisms behind the biological effects
of psychogenic stress [6, 7].
These stress-inducing paradigms must be able
to induce the activation of the hypothalamic-pituitary-
adrenal (HPA) axis through the secretion of cortisol,
and/or the sympathetic adrenal medullary (SMA) axis
with the release of noradrenaline; both hormones
physiological markers of stress [5]. More recently, the
IL6-STAT3 axis has been revealed as a pathway also in-
volved in mediating the effects of psychogenic stress,
where systemic IL-6 elevation has been associated
with stressful conditions [8], with the potential to affect
a diverse array of cellular and molecular targets [9].
Other markers of chronic stress activation also include
body weight fluctuations and changes in behavior,
including depression and feeding behavior [10]. In-
terestingly, cancer, as a form of physiological stress,
can also elicit these same responses [11].
In the context of the increasing concern regarding ani-
mal well-being in biomedical research, for psychogenic
stress research, the induction of stress makes necessary
to cause a certain level of discomfort in experimental
animals, but how much and for how long is not a settled
matter, although efforts have been made to optimize
times of induction and parameters [12]. For chronic
stress induced by restriction of movement, we have
found that a significant number of studies employ the
restraint paradigm for periods ranging from 2 to 6 h [4].
Previously, Zamora-González [13] had demonstrated
that a daily protocol of 30 min of restraint stress, that
also includes the aleatory use of the forced swim test,
as a stress inductor, and resting days to avoid habitua-
tion in animals, is enough for the activation of the HPA
axis in BALB/c mice. In this work, we applied this short-
timed stress inducing protocol to test its ability to elicit
a chronic stress response in the L5178Y-R mouse model
of lymphoma, with the aim of establishing an effective
mouse model to study cancer initiation and progression
under psychogenic stress and that is also compliant with
animal welfare concerns regarding the use of experimen-
tal animals in biomedical research.
MATERIAL AND METHODS
Animals. Female, 10–12-week-old BALB/c mice
were purchased from Harlan Laboratories S.A. de C.V.
Submitted: September 8, 2017.
*Correspondence: E-mail: diana.caballerohr@uanl.edu.mx
Abbreviations used: HPA — hypothalamic-pituitary-adrenal; NE —
norepinephrine; SAM — sympathetic adrenal medullary.
Exp Oncol 2017
39, 4, 276–280
Experimental Oncology 39, 276–280, 2017 (December) 277
(México City, Mexico) and were housed in an ani-
mal care facility at 24–26 °C with 45% humidity and
a 12 h light: 12 h dark cycle, in individually ventilated
cages with environment enrichment; water and food
were provided ad libitum. All procedures performed
in animals followed ethical standards on animal welfare
and were approved by the Institutional Animal Care and
Use Committee (CICUAL-LIV).
L5178Y-R lymphoma model. To test the influ-
ence of a chronic stress paradigm on tumor bearing
animals we used the L5178Y-R lymphoma mouse
model. This lymphoma is maintained in ascites form
in BALB/c mice by the intraperitoneal transplantation
of 1•106 L5178Y-R cells per mouse. Thirteen days
after inoculation, ascites is collected from the perito-
neal cavity of mice killed by cervical dislocation under
anesthesia. The cell suspension is then washed twice
in PBS by centrifuging at 2000 rpm for 10 min, finally
adjusting to 2•107 cells/ml in sterile PBS. To induce the
formation of a solid tumor, 0.1 ml of the cell suspension
was inoculated in the right flank of mice by intramus-
cular injection [14].
Chronic stress protocol. Animals were randomly
distributed into 4 experimental groups (n = 4): non-tumor
bearing animals in resting conditions (a) and under
stress (b); and lymphoma-bearing animals in resting
conditions (c) and under stress (d). Animals in resting
conditions remained in their cages while the stress ses-
sions were carried on. Experimental mice were subjected
to a 28 days chronic stress protocol consisting in two
stress paradigms [13]: restraint stress (30 min) and forced
swim (15 min); these were randomly applied from day 0,
as shown in Fig. 1. For restraint stress, the animals were
placed in 50 ml conical tubes with ventilation holes. A sec-
ond, larger container was employed for tumor-bearing
animals once tumor size made difficult to contain the ani-
mals in the conical tubes. For the forced swim test, a plastic
cylindrical tank, 19 cm height by 15 cm diameter, was
employed; the container was filled up to 8.5 cm from the
bottom with tap water, temperature was set at 25 °C. Mice,
one at a time, were carefully placed in the water under
constant vigilance, and after 15 min, were removed and
gently dried before being returned to the cage.
On day 4, mice were inoculated with the
L5178Y-R lymphoma, as previously described. On days
9, 16 and 23 the anhedonia test was performed. Body
weight was measured every 4 days. At the end of the
protocol, blood was collected by cardiac puncture
of mice under anesthesia, after which animals were
euthanized by cervical dislocation and tumor and liver
samples were collected and stored frozen. Blood was
centrifuged at 5000 rpm, and plasma was collected
and stored at −80 °C until analysis.
Anhedonia test. To assess behavioral disrup-
tions caused by the stress protocol, animals were
subjected to the sucrose preference test, also known
as anhedonia test. This test measures the animal’s pre-
ference for sweetened drinks, a reward, over plain
water, as an indicator of the ability to feel pleasure.
The test was carried out in the housing cages, two
drink bottles per cage, one containing 200 ml of tap
water, and the other the same volume of a 2% sucrose
solution (Jalmek Científica, S.A. de C.V.) were made
available to the animals for 48 h. Water and sucrose
solution intake were measured at 24 and 48 h. Su-
crose preference was calculated as the percentage
of sucrose solution intake over total fluid intake [15].
Before testing, the animals were habituated to having
the 2 bottles, water and sucrose solution, over a 3 day
period, switching the position of the bottles every
24 h [16], to avoid potential bias due to the rejection
of a new item by the animals.
Cytokines and hormones. Plasma levels of IL-2,
IL-4, IL-6, IFN-γ, TNF, IL-17A, and IL-10 were deter-
mined in an Accuri 6 flow cytometer using the Cy-
tometric Bead Array Mouse Th1/Th2/Th17 kit from
BD Biosciences (San Jose, CA), data was analyzed
with the FCAP Array Software V 3.0 (Soft Flow Hungary,
Ltd., Pécs, Hungary). Plasma levels of corticosterone
and norepinephrine were measured with the Corticos-
terone EIA Kit from Enzo Life Sciences (NY, USA) and
the Mouse Noradrenaline ELISA kit from MyBiosource
(San Diego, California), respectively. Insulin and leptin
were measured in a MAGPIX® system using the Mouse
Metabolic Magnetic Bead Panel from Merck Millipore.
Statistical analysis. Level of significance was as-
sessed by Student’s t test. Statistical analyses were per-
formed by using the website for statistical computation
VassarStats, freely available at http://vassarstats.net/.
RESULTS
We subjected non-tumor bearing and lymphoma-
bearing mice to a short-timed stress paradigm for
28 days (Fig. 1); we found that this chronic short stress
induction was sufficient to significantly alter some key,
but not all, immunological and metabolic parameters
evaluated. Despite being brief, the stress protocol
interfered with weight gain in non-tumor and lymphoma-
bearing mice. In Fig. 2, the weight progression of ex-
perimental animals during the experiment is shown.
By the end of the protocol, at day 28th, non-bearing
tumor animals in resting conditions showed a 9% mean
increment of their weight, meanwhile, stressed mice
not only failed to gain weight, at day 28 their mean
weight was 3% lower than on day 0 of the experiment
(p < 0.007). For lymphoma-bearing mice, weight gain
in stressed animals was half of those resting (16%),
here; the increasing weight displayed by lymphoma-
bearing animals must be attributed to tumor growth,
having the unintended effect of masking weight loss
by the animals.
Tumor progression
Day
REREFS FS FS FS FS FS FS FS FS FSRE
0 4
T AT AT AT S
9 16 23 28
RERE RE R R RRE RE RE RE RE RE RE RE RE RE
Fig. 1. Stress protocol timeline. Female BALB/c mice,
11 week-old at the beginning of the experiment, were subjected
to a chronic stress protocol combining the restraint and forced
swim paradigms for 28 days. Control animals were kept undis-
turbed in their cages. RE — restraint stress; FS — forced swim;
R — resting; T — tumor inoculation; AT — anhedonia test; S —
mice sacrifice and sample collecting
278 Experimental Oncology 39, 276–280, 2017 (December)
25
27
29
31
33
35
37
39
41
43
0 11 15 19 23 26
BA
LB
/c
m
ic
e
bo
dy
w
ei
gh
t,
g
Stress induction (day)
Non-tumor resting
Non-tumor stressed
Tumor-bearing resting
Tumor-bearing stressed
*
*
Fig. 2. Body weight evolution under chronic stress. Body
weight was measured every 3 days. Weight gain is expressed
as a percentage of the weigh at the beginning of the experiment.
Data represent mean ± SEM of 4 animals from a representative
experiment. *p < 0.05 as compared with the respective control
To evaluate the effect of chronic stress on the
L5178Y-R model, the anhedonia test was performed
twice during the experiment; for 48 h, animals were
offered the choice between two bottles, one containing
tap water and the other a 2% sucrose solution, a de-
crease in sucrose solution intake would be indicative
of depression in the animals, however, no differences
were detected among the experimental groups, find-
ing sucrose preference across the groups ranging
from 76–81%, above the 65% used as criterion for
anhedonia [16].
To test the activation of the HPA and adrenergic
system, the plasmatic levels of corticosterone and
norepinephrine were measured using ELISA. Accord-
ing to the results, the short induction of stress used
in this study failed to activate the HPA system, levels
of corticosterone remained unchanged between the
experimental groups (Table). As for the adrenergic
system, although norepinephrine levels were unaf-
fected by stress, a 240% increase was observed for
resting lymphoma-bearing animals when compared
to resting non-tumor bearing animals (p < 0.008),
suggesting that tumor burden, but not the stress
protocol tested, activates the sympathetic system
in BALB/c mice (Table).
Table. Neuroendocrine responses of female BALB/c mice to chronic stress
Mice Groups Corticosterone, pg/ml Norepinephrine, pg/ml
Non-tumor Resting 111.5 ± 8.7 131.3 ± 34.6
Stress 124.5 ± 4.7 163.8 ± 118.0
Tumor Resting 120.3 ± 6.1 315.2 ± 88.2
Stress 138.5 ± 6.8 262.8 ± 78.0
Plasma levels of Th1, Th2 and Th17 cytokines
were also evaluated. Here, we found that although
IL-4, IL-2 and TNF were detected (concentration
ranging from 8.5–10 pg/ml), no differences were
observed among the experimental groups. Unex-
pectedly, IL-6 was not detected in any experimen-
tal group. Then, according to the results, anti and
pro-inflammatory cytokines were mostly unaffected
by the short time of stress protocol tested, with one
important exception, levels of IFN-γ were significantly
elevated in lymphoma-bearing mice (40.06 ± 9 pg/ml)
when compared to non-bearing tumor animals, how-
ever lymphoma-bearing animals under chronic stress
showed 50% lower levels of plasmatic IFN-γ (p < 0.05)
(Fig. 3).
Non-tumor bearing animals showed a reduction
of 95.8% in levels of plasmatic leptin in animals under
stress (p < 0.01). In lymphoma-bearing animals, le-
vels of leptin were also lower when compared to non-
tumor bearing animals, regardless of stress induction
(Fig. 4). Although insulin was detected in non-tumor
bearing animals (236 ± 160 pg/ml), it was not detected
in non-tumor bearing animals under stress, or in rest-
ing or stressed lymphoma-bearing mice.
IF
N-
γ,
p
g/
m
l
0
10
20
30
40
50
60
Non-tumor
*
**
Tumor-bearing
BALB/c mice
Resting Stressed
Fig. 3. Plasmatic levels of IFN-γ in BALB/c mice under chronic
stress. At day 28, animals were anesthetized with ketamine/
xylazine and whole blood was collected by terminal cardiac
puncture, sample collection was performed between 16:00 and
20:00 hours. Levels of IFN-γ were measured by flow cytometry
using a cytometric bead array. Data represent mean ± SEM
of 4 animals from a representative experiment. *p < 0.05,
**p < 0.01 as compared with the respective control
Non-tumor
**
Tumor-bearing
BALB/c mice
Resting Stressed
0
500
1000
1500
2000
2500
3000
Le
pt
in
, p
g/
m
l
Fig. 4. Plasmatic levels of leptin in BALB/c mice under chronic
stress. At day 28, animals were anesthetized with ketamine/
xylazine and whole blood was collected by terminal car-
diac puncture, sample collection was performed between
16:00 and 20:00 hours. Levels of leptin were measured by mul-
tiplexed immunoassay in a MAGPIX® system. Data represent
mean ± SEM of 4 animals from a representative experiment.
**p < 0.01 as compared with the respective control
DISCUSSION
In this work, with the aim of establishing a refined
model to measure the effects of psychogenic stress
in cancer, considering the welfare of experimental ani-
mals, we measured the effects of a short-timed chronic
stress protocol in mice bearing the L5178Y-R lympho-
ma. We found that although not all parameters were
affected, some were distinctly altered by the stress
protocol; with some of the observed changes suggest-
ing a disruption in the ability to maintain body weight
that is potentially being driven by adrenergic activation.
Experimental Oncology 39, 276–280, 2017 (December) 279
Body weight is a useful and inexpensive parameter
to assess the effect of stress on living beings under
various conditions. In this work, under stress, the body
weight of non-tumor bearing animals was significantly
lower than in resting non-tumor bearing mice, this dis-
ruption could also be observed in lymphoma-bearing
mice. Weight loss is an indication of a compromised
energy metabolism in the animals, it can also be the
result of behavioral changes, such as decreasing
food intake, however we have no data to support this
hypothesis and the results of the anhedonia test (data
not shown) do not suggest that this short-timed stress
induction paradigm is enough to change the behavior
of animals.
The sustained activation of both the HPA axis
and adrenergic pathways during chronic stress may
trigger adaptive thermogenesis with the mobiliza-
tion of the body energy stores leading to weight loss.
Under stress, changes in feeding behavior could also
be involved in weight loss. These changes may be at-
tributed to HPA activation, however, according to our
results, the protocol tested was not able to activate the
HPA axis; levels of corticosterone were not different
between resting and stressed animals. This contradicts
the findings of Zamora-González et al. [13] since they
reported the elevation of corticosterone in animals
subjected to the same stress protocol. Given that they
used mice of the same strain, sex, and age, these
contradicting results may be explained by the time
of the day the sample was collected. Overall, there
is ample controversy regarding HPA axis activation
as an indicator of stress induction, since a signifi-
cant number of studies have failed to find alterations
in corticosterone levels in animals under restraint and
social stress [17].
Another potential contributor to weight loss is the
elevation of peripheral IL-6, a pro-inflammatory cy-
tokine that has been linked to the induction of un-
coupling-1 protein leading to brown tissue activation
and weight loss [18], however, as previously stated
in the results section, this cytokine was mostly absent
in plasma in experimental animals, regardless of treat-
ment. As for adrenergic activation, lymphoma-bearing
animals showed elevated plasma levels of norepi-
nephrine, and although non-tumor bearing animals
under stress also showed an elevation when compared
to resting animals, it was not statistically significant.
Weight decrease in non-tumor bearing animals under
stress, may be in part due to adrenergic activation.
The analysis of plasmatic levels of leptin and in-
sulin supports the view of a disrupted metabolism
in the animals, caused not only by stress but also
by tumor burden, that may be involved in weight
maintenance. In non-tumor bearing animals under
stress, levels of leptin were significantly lower, 95.8%,
than in resting animals. Levels of leptin were not dif-
ferent in L5178Y-R lymphoma-bearing mice under
stress when compared with resting animals. It could
be expected that changes in levels of leptin, a satiety
messenger, to be accompanied by changes in food
intake behavior, playing a role in weight maintenance,
something that needs to be assessed. Insulin was ab-
sent in non-tumor bearing animals under stress, and
in lymphoma-bearing mice, under resting and stress
conditions as well.
In regard to IFN-γ, it was evident that tumor burden
causes an elevation in plasmatic IFN-γ in the animals;
this change is consistent with an antitumoral response
mounted by the host. However, such a response
was reduced in lymphoma-bearing mice subjected
to stress by half. Previously, it has been demonstrated
that acute and chronic stress by restraint or immo-
bilization induces a decrease in INF-γ plasma levels
in rats [19, 20] and in a mouse model of contact hyper-
sensitivity also [21]. This effect has also been observed
in pre-clinical models of cancer, where decreasing
IFN-γ expression contributes to tumor progression
in mouse models of lymphoma, colorectal cancer and
melanoma [22–24]. In these studies, the daily protocol
for stress induction ranged from 2 to 4 h when restraint
or immobilization was used, some other authors used
psychosocial paradigms for up to 24 h, whereas in the
present study, 30 min of daily stress were sufficient
to find a significant effect after 28 days. The relevancy
of IFN-γ suppression by stress during cancer progres-
sion should not be underestimated; however, recent
findings shed light on a potential additional target
of this stress-induced immune dysfunction, since it has
been shown that IFN-γ supports social behavior and
neuronal connectivity [25]. Moreover, in mice knock-
out for IFN-γ, attenuation of the neuroendocrine and
immune responses to chronic stress and disturbed
spatial recognition memory in the basal state have
been observed [26, 27]. These studies suggest a role
for IFN-γ in mediating stress responses.
In conclusion, our results show that the daily induc-
tion in non-tumor and lymphoma bearing BALB/c mice
of 30 min of restraint stress, with intercalated days
of forced swim and days to rest, disrupts metabolism
as shown by decreased weight, leptin, and insulin, this
despite an absence of HPA axis activation or increasing
IL-6 levels in plasma, with the potential involvement
of adrenergic activation according to norepinephrine
plasma levels. The protocol was also able to suppress
the IFN-γ response of lymphoma-bearing animals,
a dysfunction that many studies show to be relevant
to understand the relationships between neuroen-
docrine and immune parameters during health and
disease.
ACKNOWLEDGMENTS
We are grateful to the Laboratory of Immunology
and Virology, FCB, UANL for their support for this
study. This work was also supported by grant CN362-
15 04 from Programa de Investigación Científica y Tec-
nológica (PAICYT), Universidad Autónoma de Nuevo
León (UANL), México to DCH.
DISCLOSURE
The authors have no conflicts of interest to disclose.
280 Experimental Oncology 39, 276–280, 2017 (December)
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