Immune based cancer therapy has become an exciting field
for the discovery of novel treatments and new uses for known
active substances [1-3]. Some efforts have been directed at
using chemically described compounds to elicit a particular
immune response such as interleukin modulation . Other
approaches have begun utilizing the immune response as a
direct effector against cancer, by modifications to the immune
response [5,6], or by therapy with cellular preparations .
Some groups have used small molecules as adjuvants in immune
response therapy; among these the imidazoquinoline
family has demonstrated an increase in cytokine production
. Another example from this family is imiquimod; this
compound has been successfully used against squamous cell
carcinoma causing apoptosis in a dose-dependent manner
causing activation of the intrinsic apoptotic pathway . In
this study we report the immunomodulatory responses of two
unnatural alkaloids 3-amino-7-benzylbenzimidazo[3,2-a]
quinolinium chloride (ABQ-48: NSC D-763307) and 3-nitro-
7-benzylbenzimidazo[3,2-a]quinolinium chloride (NBQ-48:
NSC D -763303). The included NSC D number relates to the
NCI designation as presented in Cox et al. .
These compounds have a planar heteroaromatic system characterized
by a quaternized nitrogen and a fused benzimidazole
(Figure 1). Both compounds are structurally related to naturally
occurring substances such as ellipticine (from Ochrosia)
and berberine (from Berberis). Ellipticine has been studied
since the 1959 and has shown a variety of biological activities
such as DNA interaction, anti-cancer activity and mitochondrial
uptake . Berberine has shown similar activities
including anti cancer activity through mitochondria interactions
. Our previous work on other compounds from this
family of benzazolo[3,2-a]quinolinium salt (BQS) compounds
has demonstrated diverse biological activities such as growth
inhibition; apoptosis induction, and DNA adduct formation in
cancer types such as epidermoid carcinoma [13-15].
In this report, we use an in vitro model to show the immunomodulatory
effects of ABQ-48 and NBQ-48. Specifically,
we show these compounds to stimulate cytokine release when
mouse splenocytes are incubated with serial dilutions of these
agents. Our data shows the expression of G-CSF, IL-2, IL-6,
and, IFN-γ to be higher after in vitro stimulation with ABQ
48 or NBQ-48, compared to non-stimulated cultures. Additional
experiments will be performed to study how the immunomodulatory
activity of ABQ-48 and NBQ-48 could be used
as a resource in the immune activation against cancer.
Materials and Methods
3-amino-7-benzylbenzimidazo[3,2-a]quinol i n i -
um chloride (ABQ-48: NSC D-763307) and 3 nitro-
7-benzylbenzimidazo[3,2-a]quinolinium chloride (NBQ-48:
NSC D -763303) were prepared as described by Cox et al.
Female 4-6 week-old BALB/c mice were purchased from
Charles River (Wilmington, MA, USA). Care of the animals
was in accordance with the guidelines from The National Institutes
of Health (Bethesda, MD, USA), and the University of Puerto Rico Institutional Care and Use Committee (IACUC).
Preparation of mouse lymphocytes
Aseptically amputated spleens from anesthetized mice were
pressed through a 70 μm nylon cell strainer (BD Falcon,
Franklin Lakes, NY, USA) using a syringe plunger, to produce
a single-cell splenocyte suspension. The resulting cells were incubated
in ACK lysis buffer (Gibco, Grand Island, NY, USA),
and washed in 15 mL of RPMI media supplemented with 10%
FBS serum and Pen/Strep. Mice were humanely euthanized by
Cell culture conditions
Murine splenocytes isolated from humanely euthanized mice
were counted and their viability calculated using a Nexelom
Biosciences Cellometer Auto T4 cell counter (Lawrence, MA,
USA). Splenocytes were incubated at 2x106 cells/mL in a flatbottom
96 well plate in 100 μL of RPMI media supplemented
with 10% FBS serum and Pen/Strep, in a humidified incubator
at 37°C and 5% CO2.
Splenocytes were incubated in twofold dilutions ranging from
5 μg/mL to 0.3 μg/mL of the BQS under study for a final volume
of 200 μL. Concanavalin A (Con A, Sigma, St. Louis, MO,
USA) and culture media were used as positive and negative
controls, respectively. Plates were incubated in a humidified
incubator at 37°C in a 5% CO2 atmosphere for five days, when
the plate was centrifuged, supernatants were removed and
stored at -80°C until testing.
Evaluation of Cytokine Profile
The cytokine profile resulting after murine spleen cells were
treated with BQS was analyzed using a fluorescence-based
multiplex ELISA microarray chip, following the protocol as
indicated by the manufacturer (RayBiotech, Norcross, GA,
USA). Screened cytokines included: G-CSF, GM-CSF, IL-1a,
IL-2, IL-3, IL-5, IL-6, IL-7, IL-10, IL-12p70, IL-13, IL-15, IL-
17, IL-21, IL-23, IFN-γ, and TNF-α.
Cytokine-profile determination shows data from experiments
that were repeated in triplicates. The immunomodulatory
activities produced from each cytokine are presented as
the mean ± standard error of the mean (SEM). The statistical
significance of differences among cytokines was determined
by One-way ANOVA, followed by the Tukey’s test, using the
GraphPad Prism statistical software (La Jolla, CA, USA). A p
value of less than 0.05 was considered significant.
Immuno-modulatory profile of ABQ-48 and
The following cytokines were analyzed in this experiment:
G-CSF, GM-CSF, IL-1a, IL-2, IL-3, IL-5, IL-6, IL-7, IL-10, IL-
12p70, IL-13, IL-15, IL-17, IL-21, IL-23, IFN-γ, and TNF-α.
Expression of G-CSF, IL-2, IL-6, , and, IFN-γ was higher after
in vitro stimulation with ABQ 48 (Figure 2) or NBQ-48 (Figure 3) compared to non-stimulated cells. Interestingly, culture
conditions used for ABQ-48 and NBQ-48 stimulation of
mouse lymphocytes show a pro inflammatory cytokine profile.
These cytokines are known to have a role in the modulation of
Specifically, IL-6 was the highest cytokine released in culture
supernatants of ABQ 48 stimulated murine lymphocytes
(Figure 2), while both, IL-6 and G-CSF, were the highest
after NBQ-48-mediated stimulation (Figure 3). The titers
of IL-6 are constantly high after splenocyte activation using
both compounds, showing no dose-response correlation to the
amount of either alkaloids were used. Under these stimulation
conditions ABQ-48 induced an average of 57.02±1.40 pg/mL
of IL-6, while the average induced by NBQ-48 was 52.35±5.36
pg/mL. NBQ-48 was able to induce higher amounts of G-CSF
as compared to ABQ 48. Specifically, NBQ-48 induced an average
of 57.46±3.86 pg/mL of G CSF, while ABQ-48 induced
26.25±3.29 pg/mL of that cytokine.
As stated before, no dose-response correlation was observed
in the expression of IL-6 at the concentration ranges of ABQ-
48 and NBQ-48 used for stimulation, and in the expression
of G-CSF among the tested concentration range for NBQ-48.
Other experiments designed to test additional concentration
ranges might be necessary in order to identify the linear region
for the NBA-48- and ABQ-48-mediated release of these
cytokines. On the other hand, Figure 2 shows that ABQ-48
induced a positive dose-response trend in the production of
IFN-γ. In this case, ABQ-48-mediated release of IFN-γ ranged
from 10.6 to 21.7 pg/mL. Interestingly, a negative trend in the
release of IL-2 was observed when murine splenocytes were
stimulated with ABQ-48, ranging from 18.5 to 3.3 pg/mL.
Moreover, NBQ-48 induced higher release of IL-2 on almost
all experimental conditions compared to ABQ-48. However,
the NBQ-48-mediated release of IFN-γ was low under these
Among all tested conditions, the highest concentrations of
the expressed cytokines resulted when ABQ-48 was used to
stimulate the splenocytes with G-CSF, IL-2, IL-6, and IFN-γ
levels of 32.3, 18.5, 61.9, and 21.7 pg/mL respectively. These
were produced when splenocytes were stimulated with 0.6,
0.3, 1.2, and 5 μg/mL of ABQ 48, respectively (Table 1). Regarding
NBQ-48, the highest concentrations of G-CSF, IL 2, IL
6, and IFN-γ were 67.6, 37.2, 65.2, and 3.8 pg/mL, produced when stimulating with 0.6, 2.5, 5.0, and 0.6 μg/mL of NBQ-48,
respectively (Table 2).
These data reveals that besides the strong in vitro anti-neoplastic
activity already reported for ABQ-48 and NBQ-48, these
agents also have the capacity to induce a pro-inflammatory cytokine
profile. Additional experiments should be performed to
explore how the combination of these properties could be used
to test ABQ-48 and NBQ-48 as effective anti tumor agents
when combined with a relevant cancer biomarker.
Discussion and Conclusions
The proven capacity of ABQ-48 and NBQ-48 controlling tumor-
cell proliferation establishes these agents as promising
antineoplastic candidates with potential clinical implications.
Several antineoplastic agents used in cancer therapies are
known to induce adverse side effects like severe neutropenia
, thrombocytopenia, nausea [17,18], and susceptibility to
infections . These undesired effects have a negative impact
in the quality of life in patients. For many patients, not only
the cancer, but also the therapy itself undermines their daily
activities. In this regard, several anticancer chemotherapies
are combined with G-CSF (Filgrastim, commercially traded
as Neupogen®, or Pegfilgrastim commercially traded as Neulasta
®) to stimulate proliferation and differentiation of granulocytes
. This combination is effectively used to enhance
the quality of life of patients suffering from cancer [21,22] by
restoring the levels of neutrophils in blood [23-25].
Cytotoxic studies of ABQ-48 and NBQ-48 show these compounds
to control tumor proliferation, which until now have
been the major research focus of these two novel generated
compounds and other members of their family. However, it
is of great interest to further evaluate the observed cytokine
stimulating activities of these compounds. The results of this
study clearly demonstrate these BQS to stimulate the secretion
of G-CSF, an agent that, as stated before, is used in combination
with anticancer chemotherapy to activate the production
of neutrophils [23,26]. This combination therapy has been
shown to enhance the quality of life in cancer patients [23-25].
This study shows ABQ-48 and NBQ-48 to effectively induce
both effects: control of tumor growth and immune modulation
to produce G-CSF, among other cytokines. Therefore, ABQ-48
and NBQ-48 could induce tumor-proliferation control with
minimal side effects after stimulating proliferation and differentiation of granulocytes  and at some degree restoring the
levels of neutrophils in blood.
Moreover, in this report, NBQ-48-mediated stimulation of
murine splenocytes is shown to produce G-CSF, IL-2 and IL-6,
while ABQ-48 also mediates the release of IFN γ. These are
pro-inflammatory cytokines with a relevant role in the activation
of immune responses. This finding opens the new possibility
of using ABQ-48 and NBQ-48 as immunomodulatory
agents, with intend to activate the immune system against cancer.
However, this information will also be used in the rational
design of new antineoplastic agents capable of modulating the
immune response. These data set the bases for the development
of novel compounds with immunomodulatory capabilities.
In this regard, projects by several groups have focused on
the development of immunomodulators with intent to induce
an immune response against cancer [27,28].
Our exciting ex vivo data encourages us to test the immunomodulatory
effects of these agents in an in vivo mouse model.
We believe that it is crucial to test the effects of these agents
in a model where the whole pharmacokinetics, drug metabolism
and toxicological factors influence the immunomodulatory
responses. First, it is necessary to determine the impact
that these agents have in the in vivo proliferation of granulocytes
using a mouse model. This will tell us if these agents
could be able to restore that important compartment of the
immune system, as it is depleted during anticancer therapies.
It is important however to mention that existing cancer research
literature, provide some arguments on the effects of
certain pro-inflammatory cytokines and cancer progression.
Other correlations however, such as antitumor effects or the
prevention of chemotherapy secondary effects have also been
associated with the over expression of certain cytokines, especially
G-CSF [29,30]. For example, Maeda H. et al, 1994,
reported the antitumor effects of the combination of G-CSF
and TNF on a xenograft line of human medulloblastoma .
Also the combination of G-CSF a key regulator of neutrophil
production has been integrated to chemotherapy protocols
especially for certain types of Non Hodgkin’s Lymphoma.
Keeping this in mind and based on the obtained results,
it would be worth to test the adjuvant potential of ABQ-48
and/or NBQ-48. In this regard, the development of a vaccine
formulation consisting of these tested BQS combined with a
cancer-specific biomarker could be expected to induce a protective
antigen-specific immune response against a selected
tumor. The tumor controlling characteristics of this formulation,
together with the generation of G CSF, and the induced
immunostimulatory profile of these compounds, makes these
agents strong candidates to be tested as safe immune-modulators
in a pre-clinical cancer regimen.
Research reported in this publication was supported in part
by the following grants: Institutional Development Award
(IDeA) from the National Institute of General Medical Sciences
of the National Institutes of Health under grant number
P20 GM103475, infrastructure support or services were
provided in part by the NIMHD NIH RCMI UPR-MSC
8G12MD007600, and NIMHD NIH UCC 8G12MD007583. Also by The Puerto Rico Science, Technology and Research
Trust 2013-00021. The content is solely the responsibility of
the authors and does not necessarily represent the official
views of the National Institutes of Health.