ABSTRACT
Non-small cell lung cancer constitutes one the most frequent and lethal
forms of the disease. The antitumor peptide CIGB-552 is a new targeted
anticancer therapy which molecular mechanism is associated with the
inhibition of the transcription factor NF-kB, mediated by COMMD1 protein
stabilization. However, its pharmacological potential in combination
with chemotherapy is unknown. In this study, we examined the
antiproliferative capacity of CIGB-552 in combination with
chemotherapeutic agents in the non-small cell lung cancer cell line
NCI-H460 and we confirmed drug interactions in vivo , in a mouse
model of TC-1 lung cancer. We focus our research in the combination of
CIGB-552 and the antineoplastic agent Cisplatin (CDDP) in a concomitant
treatment. Our results demonstrate a clear synergic effect between 37.5
μM of CIGB-552 and 5 μM of CDDP under concomitant scheme, on
proliferation inhibition, cell cycle arrest, apoptosis induction and
oxidative stress response. The effect of CIGB-552 (1 mg/kg) and CDDP
(0.4 mg/kg) administrated as a combined therapy was demonstratedin vivo in the TC-1 murine model where the combination achieved
an effective antitumor response, without any deterioration signs or side
effects. These findings demonstrate the efficacy of the concomitant
combination of both drugs in preclinical studies and support the use of
this therapy in clinical trials.
Key words: NSCLC, antitumor, synergism, combined therapy,
CIGB-552, CDDP
INTRODUCTION
Cancer represents one of the most challenging diseases in XXI century.
Malignant transformation is a multistep process, where cancer cells gain
properties like immune evasion, apoptosis resistance, insensitivity to
antiproliferative signals, invasion, angiogenesis and metastasis.
(Hanahan and Weinberg 2011). The website GLOBOCAN and the International
Agency for Research on Cancer (IARC) estimated 18.1 million new cancer
cases and 9.6 million cancer deaths in 2018. In both sexes combined,
lung cancer is the most commonly diagnosed (11.6% of the total cases)
followed by female breast cancer and prostate cancer and is currently
the leading cause of cancer death (18.4% of the total cancer deaths),
closely followed in mortality by colorectal (9.2%), stomach (8.2%),
and liver cancer (8.2%). (Bray, Ferlay et al. 2018, 2020). In Cuba,
cancer is the second cause of death (24% of total deaths) just
surpassed by heart and circulatory diseases (36,8 % of the total number
of deaths) and lung cancer is still the one with more incidence and
mortality among Cuban population. (2020).
Non–small cell lung cancer (NSCLC), represents the most frequent
subtype of lung cancer with an 85% of incidence. The therapeutic
strategies include surgery by early diagnostic, chemotherapy and
radiotherapy in advanced cancer. The conventional chemotherapy protocols
for NSCLC comprise 4 to 6 cycles of platinum-based doublet chemotherapy
in first-line treatment and 6 cycles of docetaxel (or equivalent taxol
drug) as a second-line regimen. Both regimens employ unspecific
cytotoxic agents, which display numerous side effects (Wagner,
Stollenwerk et al. 2020). The toxicity associated to chemotherapy affect
the life quality of patients and nephrotoxicity, lymphopenia among other
adverse reactions limit its long-term application in cancer therapy in
addition to other negative impacts such as multidrug resistance (MDR),
mutagenicity and teratogenicity (Bordoni 2008, Tiwari, Sodani et al.
2011). The combination of chemotherapy agents with other drugs that
target specific tumor antigens or intracellular proteins such as
monoclonal antibodies, peptides or small chemical inhibitors has
demonstrated to be an efficient therapeutic strategy in NSCLC and other
types of tumor (Li, Zhao et al. 2014, Achkar, Abdulrahman et al. 2018,
Wagner, Stollenwerk et al. 2020) This attractive therapy achieves
efficacy and decreases toxicity, reducing the doses of the
antineoplastic agents without losing their effect. This can also
contribute to attenuate MDR (Tallarida 2001, Bayat Mokhtari, Homayouni
et al. 2017).
CIGB-552 is an antitumor peptide developed at the Center of Genetic
Engineering and Biotechnology (CIGB), Havana, Cuba. Vallespi and her
research group have demonstrated its cell penetrating capacity and
proliferation inhibition of human and murine cancer cells in
vitro (Fernández Massó, Oliva Argüelles et al. 2013, Astrada, Fernández
Massó et al. 2018). Likewise, CIGB-552 elicits tumor regression in
vivo , in syngeneic and xenograft mice models (Vallespí, Pimentel et al.
2014) as well as the stabilization of the disease and therapy
improvement in pet dogs with spontaneous cancer (Vallespi, Rodriguez et
al. 2017). The molecular studies of this peptide have shown inhibitory
effects on NF-kB pathway through
the stabilization and accumulation of the Copper Metabolism Mur 1 Domain
Containing Protein 1 (COMMD1). This effect induces the ubiquitination
and degradation of the NF-kB subunit RelA. In addition, CIGB-552 induces
oxidative stress in tumor cells by the inhibition of the enzyme
Superoxide-dismutase 1 (SOD1), causing lipid and protein peroxidation
(Fernández Massó, Oliva Argüelles et al. 2013). These findings
complemented by additional evidence in proteomic studies (de
Villavicencio-Díaz, Gómez et al. 2015) suggest that the combination of
CIGB-552 and conventional chemotherapeutic drugs such as
Cisplatin
(CDDP) or
Paclitaxel
could be attractive in preclinical and clinical settings. Therefore, in
this work, we evaluated for the first time the combination of CIGB-552
with these antineoplastic agents widely use in lung cancer therapy, in
the human NSCLC cell line NCI-H460. We explored two combination
settings: pre-treatment and concomitant in order to select the optimal
combination scenery to use in clinical trials, based on the estimation
of the combination index (CI) (Chou 2006). All the interactions were
classified as synergistic, additive or antagonistic and their
proliferation inhibition capacity was compared. The best combination was
evaluated in terms of its effect on cell cycle progression, apoptosis
induction and oxidative stress triggering. Furthermore, the potential
benefit of the best combination and therapeutic scheme was corroboratedin vivo in a mouse model of lung cancer.
MATERIALS AND METHODS
Reagents and Chemicals
All regents and chemical substances used in this study were purchase
from Sigma-Aldrich. Culture media, fetal bovine serum (FBS), and cell
culture material were obtained from Life Technologies (USA), GE
Healthcare, and Greiner. All reagents for peptide synthesis were of
synthesis grade. Reagents for chromatography were of high-performance
liquid chromatography (HPLC) grade.
Antineoplastic drugs
The clinical grade chemotherapeutic drugs Cisplatin (CDDP), and
Paclitaxel (Drug Research and Development Center, Havana, Cuba) were
kindly provided by the Oncology Service of the National Institute of
Oncology and Radiobiology (Havana, Cuba). Both were dissolved in
buffered saline solution (PBS) for in vitro experiments and CDDP
was also prepared in PBS for its administration in vivo .
CIGB-552
Peptide CIGB-552 was synthesized on a solid phase and purified by
reverse-phase-HPLC to >95% purity on an
acetonitrile/H2O trifuoracetic acid gradient and
confirmed by ion-spray mass spectrometry (Micromass, Manchester, UK).
The synthesis of the peptide in solid phase was performed using the
Fmoc/t-Bu chemistry. The linking is direct to the N-terminus of the
peptide; there are no additional residues. Lyophilized peptide was
reconstituted in phosphate buffered saline (PBS) for use in vitroand in vivo .
Cell lines
NCI-H460 (human non-small cell lung carcinoma), MRC-5 cells (human
embryonic lung fibroblasts) and TC-1 (murine lung epithelial cells
transfected with VPH-16) were acquired from the ATCC and cultured in
RPMI 1640 (for NCI-H460 and TC-1) and DMEM (for MRC-5), supplemented
with Glutamax and 10% (v/v) FBS according to the recommendations of the
supplier. Cells were maintained in a 5% CO2 atmosphere
at 37°C in incubator. Cells were cultured for no longer than 10–15
passages.
Antiproliferative assays
The effect of CIGB-552, CDDP and
Paclitaxel on cell proliferation was then evaluated by a sulforhodamine
B (SRB)-based assay according to the method described by the National
Cancer Institute (Boyd 1997). NCI-H460 Cells were seeded at a density of
4x104 cells per well in a 96-well plate (Costar, USA).
Then after 24 hours they were incubated with the products (10, 40 and
100 µM of CIGB-552; 0.05, 5 and 50 µM of CDDP and 0.2, 20 and 200 µM of
Paclitaxel) for 48 hours and the viability relative to untreated cells
was measured by the SRB method. The percentage of cell proliferation
inhibition was determined using CalcuSyn software (Version 2.1; Biosoft,
Cambridge, UK). The assay was performed twice with three replicas for
each concentration.
In vitro drug combination study
The drug combination study was carried out in 96-well plates seeded with
NCI-H460 cells (4x104 cells per well) and the cell
viability was measured after 48 hours of treatment using the (SRB)-based
assay as we described above. Cells were treated with different
concentrations of CIGB-552 (300 - 9.37 μM) and the antineoplastic drugs
(500 - 5x10-3 μM of CDDP or 2 -
2x10-4 μM of Paclitaxel) according to a Latin square
design (Chou 2006). The assay was performed under two different
treatment schemes: concomitant (CIGB-552 and the chemotherapeutic agent
were added at the same time) and pre-treatment (cells were pre-incubated
with CIGB-552 for 5 hours, then it was eliminated from culture media and
the cytostatic drug was added). The effect on cell proliferation was
determined relative to untreated cells in two independent experiments.
The results were analyzed with
CalcuSyn software to determine the
type of interaction (synergism, additivity or antagonism), according to
the obtained combination index (CI) values. The software also calculated
the dose reduction index (DRI) and the fraction affected (Fa) which is
related with the magnitude of effect for each combination. The 2D
interaction maps with color-coded surfaces were created using Matlab®
R2012a software based on the CI and Fa values.
Cell cycle analysis
The cell cultures were incubated with CIGB-552
(37.5 µM), CDDP
(5 µM) and their combination for
12 and 24 hours. Subsequently, the cells were collected by
trypsinization, washed and fixed with ice‑cold methanol/acetone (4:1) at
4˚C for 1 hour. Cells were then stained by incubation for 20 min at 37˚C
with a solution of 100 mg/ml PI and 10 mg/ml of DNase‑free RNase.
Stained cells were analyzed on a Becton Dickinson FACSCalibur cytometer
using the cell cycle analyzer from CellQuest software. Prior to fitting
the DNA distribution to a diploid DNA content for cell cycle profiling,
cellular debris and doublets were properly excluded by gating out in
FL3-A vs. FL3-W two-parameter dot plots.
Western Blot of apoptosis related proteins
NCI-H460 cells were treated with CIGB-552 (37.5 µM), CDDP (5 µM) and
both drugs combined, as well as 1 μM of Staurosporin (STS) as positive
control. Cell fractioning was performed as described previously (Thoms,
Loveridge et al. 2010) using Lysis Buffer (Cell Signaling, USA) and
protein inhibitor cocktail (Roche, USA), according to the instructions
of the supplier. Proteins in whole cell extracts (30 µg) were resolved
by electrophoresis on 12.5% and 15% polyacrylamide gels (Bio-Rad
technology, USA) and analyzed according to the western blotting
technique previously described (Burnette 1981). Briefly, the proteins
were transferred to a nitrocellulose membrane (pore size 0.45 mm) and
incubated with the appropriate primary antibody (1:500-1:1000 dilution).
After incubation with peroxidase-conjugated secondary antibody (1:
2000), protein specific bands were visualized using chemiluminescence
reagents followed by exposure to standard X-ray films.
Annexin V/ Propidium iodide double staining
Cells in early and late stages of apoptosis were detected with an
Annexin V-FITC apoptosis detection kit from Sigma (041M4083). NCI-H460
cells (1x105cells per well) were treated with CIGB-552
(37.5 µM), CDDP (5 µM) or the combination in 12-well plates (Costar,
USA) and incubated for 48 hours prior to analysis. STS (1 μM) was used
as positive control of apoptosis induction. Then, cells were trysinazed,
collected and resuspended in 1x binding buffer. To 100 μL of cell
suspension, 5 μL of Annexin V-FITC and 10 μL propidium iodide (PI) were
added and incubated for 10 min at room temperature prior to analysis.
Samples were analyzed (20,000 events) using
the
Becton Dickinson FACSCalibur
instrument and CellQuest software. Cells that were positive for Annexin
V-FITC alone (early apoptosis) and Annexin V-FITC and PI (late
apoptosis) were counted.
Superoxide anion
accumulation assays
The detection of superoxide anion formation as a measure of oxidative
stress induction was determined as previously described (Wojtala, Bonora
et al. 2014). NCI-H460 and MRC-5 cells were seeded at a density of
6x103 and 1x104 cells per well,
respectively, in 96-well plates. After 24 hours they were treated with
CIGB-552 (37.5 µM), CDDP (5 µM) and the combination for 1 hour.
Hydrogen peroxide
(H2O2) 2.5 μM was used as positive
control. Then cells were incubated with
10 μM of Hydroethidine (HE) for 1
hour to visualize the superoxide anion formation. The fluorescence was
detected and measured using confocal microscopy (Inverted microscope
Olympus, Japan). The images acquired were analyzed with the software
ImageJ 1.41 to quantify the fluorescence intensity as fold of control
for all treatments. We processed images from two independent
experiments.
To confirm the induction of oxidative stress in NCI-H460 cells, we
perform a flow cytometry-based assay for HE detection in treated cells
according to the method described by Walrand and colleagues (Walrand,
Valeix et al. 2003). Briefly, we seeded 1 x 106 cells
per well and treated with the products alone and combined for 12 hours.
Untreated cells and cells incubated with 2.5 μM of Hydrogen peroxide
(H2O2) for 12 hours were the negative
and positive control, respectively. Cells were mechanically detached in
cold and cell suspensions were obtained by centrifugation at 300 x g for
5 minutes at room temperature. Pellets were re-suspended in PBS 1X and
incubated with HE (10 μM) for 30 minutes at 37°C protected from light.
Then cells were analyzed in the Becton Dickinson FACSCalibur cytometer
and corresponding CellQuest software. A total of 2x104events were analyzed and HE positive cells were quantified. We
established forward and side scatter gates from negative control cells
to exclude debris and cellular aggregates.
In vivo drug interaction experiments
C57/BL6 female mice of 8 weeks old and 18-20 g (a total of 40 animals)
were randomly divided into 4 groups of 10 animals each. Mice were
injected with 5x104 cells per animal of the murine
tumor cell line TC-1 on the right flank, according to the procedures
reported for this animal model (Tanaka, Delong et al. 2005). When tumors
reached 70-90 mm3 we start the administration of the
products at the indicated doses. Group 1 received saline solution (PBS
1x) by subcutaneous route three times a week during three weeks (control
group). Group 2 was subcutaneously administered with 1 mg/kg of
CIGB-552, three times a week during three weeks. Group 3 received 0.4
mg/kg of CDDP by intraperitoneal route three times a week only during
the first week of administration. Group 4 was administered with a
combination of both drugs: CIGB-552 (1 mg/kg) subcutaneously three times
a week during three weeks and CDDP (0.4 mg/kg) intraperitoneally three
times a week during the first week. Animals were weighted once a week
since the beginning of the experiment. Tumors were measured three times
a week with a caliper and their volumes were calculated according to the
following formula: volume = length x width2/2.
Survival rate was daily registered during the experiment and only when
tumor volumes reached 2,000 mm3, the animals were
sacrificed due to ethical considerations. Mice were maintained under
pathogen-free conditions and all the procedures were performed in
accordance with the recommendations for the proper use and care of
laboratory animals at the Center for Genetic Engineering and
Biotechnology (Havana, Cuba). This animal study complies with all the
international requirements and is according the National Institutes of
Health guide for care and use of laboratory animals.
Statistical analysis
The drug-drug interactions were determined and validated using the
software CalcuSyn version 2.0, (1997, Biosoft, EUA). The percentages of
cell proliferation inhibition, AV + AV/PI positive cells, HE
fluorescence intensity (fold of control) and number of affected animals
from in vivo experiments were compared between the combination
and the individual treatments using
one way ANOVA and Dunnet post
test. The number of HE positive cells in percent determined using flow
cytometry, as well as tumor volumes from in vivo experiments were
compared between the combination and the individual treatments by
unpaired T tests. The statistical significance of differences in
survival rates was determined by log-rank test p*<0,05. All statistical analysis were done using the software
GraphPad Prism 7.
Nomenclature of Targets and Ligands
Key protein targets and ligands in this article are hyperlinked to
corresponding entries in http://www.guidetopharmacology.org, the common
portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et
al., 2018), and are permanently archived in the Concise Guide to
PHARMACOLOGY 2019/20 (Alexander et al., 2019).
RESULTS
CIGB-552 and cytostatic drugs are synergic in human lung
cancer cells
First, we evaluated and compared the cytotoxicity of our anticancer
peptide CIGB-552 in combination with cytostatic drugs commonly used in
chemotherapy of lung cancer, such as CDDP and Paclitaxel, in the human
lung cancer cell line NCI-H460. We selected this cell line based on
previous results obtained in our lab that reveal a major
cytotoxic potential in NCI-H460
cells in comparison to other human lung cancer cell lines (data not
shown). In addition, the molecular mechanism of CIGB-552 has been fully
characterized by us in NCI-H460 cells (Fernandez J et al., 2013). We
have previously obtained and corroborated the cytotoxic effect of
CIGB-552, CDDP and Paclitaxel in this cell line, with
IC50 values of 44.6, 1.99 and 0.01 µM, respectively
(data not shown). Based on these evidences we decided to evaluate the
antiproliferative capacity of the peptide and both cytostatic drugs
separately and combined in NCI-H460 cells, testing different
concentrations below and above the IC50 as it is
recommended. All treatments were able to inhibit cell proliferation in a
concentration dependent manner, but this effect was more potent
combining the peptide with the cytostatic drugs, particularly at lower
concentrations where the effects of both products is not overlapped
(Figure 1A and B ). To corroborate this possible
synergic effect and to identify other pharmacological interactions, the
antiproliferative capacity of CIGB-552 combined with CDDP and Paclitaxel
in a wider range of concentrations was evaluated in the lung cancer cell
line NCI-H460, by an in vitro drug combination assay. We treated
the cells with CIGB-552 and both cytostatic drugs alone and in
combination, using two different settings: concomitant and
pre-treatment. The results were analyzed with the software Calcusyn to
determine the significance or grade of combination (synergism,
additivity and antagonism) based on two main parameters: combination
index (CI) and dose reduction index (DRI) (Table 1 ).
In the concomitant setting, we observed antagonism between 300 µM of
CDDP and 150 µM of CIGB-552; however, this concentration of the peptide
interacted with 50 µM of CDDP, producing an additive effect with 95% of
cell proliferation inhibition. Interestingly, the synergism was observed
in concentrations below 150 µM of CIGB-552 and 50 µM of CDDP where the
cell growth inhibition is still 95% in the range of 37.5 µM to 9.37 µM
of CIGB-552. The synergism is potent in 0.5 - 5 µM of CDDP and 18.75 -
9.37 µM of CIGB-552, with CI values between 0.7 and 0.1 (Figure
1C ). Compared with the concomitant, pre-treatment analysis showed
antagonic effect in maximal and minimal concentrations of both drugs,
being potent in two zones: 500 µM of CDDP with the full concentration
range of CIGB-552 and also 0.5 - 5 µM of CDDP with 18.75 - 9.37 µM of
CIGB-552, demonstrating that the surface of synergism is reduced under
this treatment scheme. Therefore, the concomitant scenario is better to
combine these drugs in NCI-H460 cells.
The combination of CIGB-552 with Paclitaxel showed a different behavior.
In concomitant scheme we observed additivity between various
concentrations of Paclitaxel and 150 - 75 µM of CIGB-552. On the other
hand, the synergic effect was found in concentrations lower than 75 µM
of CIGB-552 and 0.2 µM of Paclitaxel, with 90% of cell proliferation
inhibition. Specifically, the range 0.002 - 0.0237 µM of Paclitaxel with
the full range of CIGB-552 achieves a potent synergism (CI = 0.3 - 0.1)
with 85% of cell death. As occurs with de combination of CIGB-552 and
CDDP, the pre-treatment setting reduced the synergism surface to the
area of 0.002 - 0.02 µM of Paclitaxel with concentrations of CIGB-552
under 150 µM (Figure 1C ). In addition, the effect on cell
growth inhibition was minor in comparison to the concomitant design.
These results show a clear synergic effect for the peptide with both
chemotherapeutic drugs demonstrating a major surface of synergism under
the concomitant combination respect to pre-treatment condition. Another
aim of this analysis is to predict a reduction of toxicity using the DRI
value, especially for drugs such as cytostatics due to their adverse
effect during cancer treatment. The DRI of CDDP and Paclitaxel in
combination with CIGB-552 is attractive and could be beneficial for
chemotherapy, particularly at low concentrations (DRI > 1)
(Table 1 ). According to this data, by using CIGB-552 as
adjuvant treatment the doses of CDDP could be reduced from 7 to 11 times
in a combined therapy maintaining a proliferation inhibition around
80-90%, whereas Paclitaxel doses could be reduced up to 13 times, but
achieving a 60% of proliferation inhibition. As is suggested by these
results, the combinations studied could help to reduce the doses of CDDP
and Paclitaxel currently used in the clinical practice, keeping the
efficacy and synergism observed.
From these experimental data we also conclude that CIGB-552 combined
with CDDP exhibits a higher synergism index and a greater cell growth
inhibition compared to Paclitaxel in several concentrations evaluated.
Also, the synergism observed was better in surface and inhibition under
concomitant treatment. For those reasons, we selected the combination of
CIGB-552 and CDDP in concomitant scheme to continue the study.
Interestingly, the major inhibition of cell growth and the higher
synergism were obtained at concentrations below the IC50of CIGB-552 (37.5 µM) and above the IC50 of CDDP (5 µM).
According to this, we decided to evaluate the effect of both drugs on
subsequent experiments at these particular concentrations.
The combination of CIGB-552 and CDDP affects cell cycle
progression
CIGB-552 and CDDP inhibited the proliferation of NCI-H460 cells and both
products combined reached a higher effect compared with their individual
inhibition. According to this, we evaluate their capacity to interfere
with cell cycle progression in NCI-H460 cells at 12 and 24 hours of
exposure, assessing the DNA content and the cell cycle phases with
Propidium Iodide (PI) measured by flow cytometry. CIGB-552, CDDP and
their combination affected cell cycle progression of NCI-H460 cells in a
time dependent manner (Figure 2 ). CIGB-552 induced cell cycle
arrest at G2/M phase (20.95%) from 12 hours of treatment compared to
control (untreated cells) and this effect was maintained after 24 hours
of exposure (20.76%). On the other hand, CDDP strongly modified the
cell cycle profile of NCI-H460 cells, inducing cell death (2.58% at 12
hours and up to 10.57% at 24 hours) and causing a clear cell cycle
arrest at G2/M phase (47.82% and 53.40% at 12 and 24 hours,
respectively). Interestingly, both drugs together induced a mixed cell
cycle profile which results from the combination of their individual
effects causing G2/M arrest (19.67% in 12 hours and 21.12% at 24
hours) and potentiating cell death, particularly at 12 h of incubation
(Figure 2A ).
CIGB-552/CDDP combination activates the apoptotic pathway
The combination of CIGB-552 and CDDP showed an increased capacity to
inhibit the proliferation of NCI-H460 cells promoting cell death and
causing perturbations in cell cycle progression, so we decided to
explore the ability of both products combined to induce apoptosis in
this cell line. For this purpose, we evaluated the effect of CIGB-552
and CDDP separately and combined on the activation of the apoptotic
pathway. First, in order of explore at a molecular level the effect of
the combination on apoptosis-related proteins we performed a western
blot analysis in NCI-H460 cells, to study whether the cell death
observed is related to the activation of
Caspase 3 (effector) and
Caspase 8 and
Caspase 9 (initiators), as well as
the cleavage of PARP, a well-known apoptosis marker. We treated the
cells with CIGB-552, CDDP and the combination of both for 12 and 24
hours. We used 1 μM of Staurosporin (STS) as positive control. InFigure 3A , we show how the Pro-Caspase 3 decreases in the
combination treatment after 24 hours of exposure respect to CIGB-552 and
CDDP, indicating the formation of active Caspase 3. At 12 hours, the
cleavage of Caspase 8 was observed in STS treatment solely, but after 24
hours, we detected the active form of Caspase 8 (41/43 kDa) in all
conditions evaluated, with a major accumulation in the combined
treatment. In the case of Caspase 9, the band corresponding to the
active form (37/35 kDa) was detected at 12 hours, and this band was more
intense in the combination. However, after 24 hours of exposure we found
no differences in the cleavage of this protein between CDDP and the
combination. Regarding the cleavage of PARP protein as a general marker
of apoptosis it was observed since 12 to 24 hours of exposure in all
treatments and the band is particularly strong after 12 hours of
incubation with CIGB-552/CDDP combination. Interestingly, after 24 hours
of treatment with the combination, the cleavage of PARP was extensive
and similar to STS. These results indicate the activation of Caspases 3,
8 and 9 as well as the cleavage of PARP by the combination of CIGB-552
and CDDP, demonstrating the stimulation of the apoptotic pathway.
Next, we decided to confirm apoptosis induction in NCI-H460 cells by the
Annexin V/Propidium iodide (AV/PI) double staining, evaluated by flow
cytometry. Cancer cells were incubated with CIGB-552 and CDDP in
monotherapy and combination during 48 hours. We also used STS (1 μM) as
positive control. All treatments increased the population of cells
stained with AV (early apoptosis) and AV/PI (late apoptosis) respect to
non-treated cells. In particular, the detection of AV/PI positive cells
was higher for the combined treatment in comparison to CIGB-552 and CDDP
alone (Figure 3B ). We graphed the percentage of AV + AV/PI
positive cells for a better understanding. The effect of the combination
reaches a 62.7 % of stained cells respect to 20.4 % and 34.5 % with
CIGB-552 and CDDP, respectively (Figure 3C ).
Altogether, these results demonstrate that the combination of CIGB-552
and CDDP have a negative effect on the survival of NCI-H460 lung cancer
cells inducing apoptosis as the mechanism of cell death. The effects
exerted by the combination are more potent compared to the drugs alone
confirming the synergic interaction between them.
Synergic antitumor effect of CIGB-552/CDDP combination is
mediated by Oxidative Stress activation
The molecular mechanism of CIGB-552 peptide in lung cancer cells is
related with the induction of oxidative stress. CDDP in addition to its
DNA intercalating activity also promotes the formation of Reactive
Oxygen Species (ROS) and oxidative damage in tumor cells. For that
reason, we expected a connection between the antitumor mechanism of the
combination and the cell redox balance. According to this, we evaluated
the formation of superoxide radical (O2•−) in NCI-H460 cells after
exposure to the products in study. Cells were incubated with CIGB-552,
CDDP and the combination during 1 hour and then they were stained with
Hydroethidine (HE) to visualize the formation of superoxide anion. A 1
hour exposure to 2.5 μM of
hydrogen peroxide (H2O2) was used as
positive control for the experiment. Both, the treatment with CIGB-552
and the combination promoted the increase of fluorescence in treated
cells, demonstrating the accumulation of O2•−, in contrast to CDDP that
showed a minor effect (Figure 4A ). Comparing the mean
fluorescence intensity of HE for the different treatments we can observe
that CIGB-552 produced a 1.8 fold increase in comparison to control
(untreated cells) and combined with CDDP the fold increase is up to 2.8,
which is statistically different from their individual effects
(Figure 4B ). To evaluate the selectivity of ROS induction as
antitumor mechanism we tested the same concentrations of CDDP and
CIGB-552 alone and combined during 1 hour of exposure in human lung
fibroblasts (MRC-5 cell line). As we expected the evaluated products do
not stimulate O2•− accumulation in
these cells (Figure 4C ), suggesting that CIGB-552 and the
combination induce this mechanism selectively in tumor cells in
comparison to non-transformed cells from the same histologic
localization. Then, in order to corroborate the ROS production in
NCI-H460 cells in response to our products and to verify this effect
after a prolonged exposure, we determined the percentage of HE positive
cells after 12 hours of incubation with CIGB-552, CDDP and the
combination of both, using flow cytometry. We used
H2O2 (2.5 μM) as positive control. The
accumulation of O2•− was detected after 12 hours of incubation in cells
treated with CIGB-552 (5% of positive cells) and interestingly in
CDDP-treated cells in a similar extent (5.3 % of positive cells).
Comparing this effect with the HE fluorescence obtained after 1 hour of
exposure, we confirm our hypothesis that CIGB-552 induces oxidative
stress as an early event that is maintained, whereas CDDP activates ROS
later in time; but in both cases this mechanism leads to apoptosis
induction and cell death. In line with this, the combined treatment
showed a significant increase in the percentage of HE positive cells
(13.8%), demonstrating synergy in the induction of oxidative stress
mediated by the products (Figure 4D ).
There is an increase on superoxide radicals when cells are treated with
CIGB-552 and this effect is stronger in combination with CDDP, as occurs
with the other parameters included in our study. In addition, not
CIGB-552 or the combination promote the generation of superoxide anion
in normal cells. Thus, the combination could help to decrease the
non-specific toxicity promoted by Cisplatin, keeping the increase of ROS
as a selective mechanism of cytotoxicity in tumor cells.
CIGB-552 and CDDP are synergic in vivo in a mouse model
of lung cancer
The combination of CIGB-552 and CDDP has shown synergistic effect in
NCI-H460 cells inducing apoptosis and oxidative stress. However, thein vivo antitumor efficacy of both drugs combined in lung cancer
animal models is still unknown. We have previously established the
pharmacokinetic profile of CIGB-552 in BALB/c mice and validated its
antitumor activity in syngeneic and xenograft mouse models of colon
cancer (Vallespí, Pimentel et al. 2014). Subcutaneous administration of
0.72 or 1.44 mg/kg of CIGB-552 was able to inhibit tumor growth and
improve survival rate without toxicity signs or body weight loss in
comparison to a reference drug such as Oxaliplatin. In line with these
previous results, we decided to explore the effects of the systemic
administration of CIGB-552 and CDDP alone and as a combined therapy in a
syngeneic mouse model of TC-1 lung cancer. First, we analyzed the
antiproliferative capacity, in vitro drug interactions and the
oxidative stress induction promoted by CIGB-552 and CDDP in the mouse
tumor cell line TC-1 (murine lung epithelial cells transfected with
VPH-16) under the same conditions of NCI-H460 cells. In this murine cell
line, CIGB-552, CDDP and the combination inhibited cell proliferation,
displaying synergism in a similar concentration range compared to
NCI-H460 cells and increasing the accumulation of superoxide radicals
(Supplementary Figure 1S ). Then we generated the syngeneic
mouse model of lung cancer by subcutaneous implantation of TC-1 cells in
C57/BL6 mice, to evaluate the
antitumor activity of CIGB-552/CDDP combination in vivo . We based
on our previous results of CIGB-552 pharmacokinetics and antitumor
efficacy in colon cancer models to select the dose and administration
route, following the experimental design described in materials and
methods (section 2).
The administration of CIGB-552 and CDDP on tumor-bearing mice led to a
significant reduction of tumor growth compared with the group treated
with saline solution (p < 0,05) (Figure 5A and B ). In
addition, mice treated with the combination exhibited significantly less
tumor volume than those only administered with CIGB-552 or CDDP
(p
< 0,05). The treatment
was also safe and tolerable for mice included in the study, as we could
corroborate by monitoring the body weight and possible toxicity signs of
treated animals. CIGB-552 and the combination practically did not affect
the life quality, whereas animals administered with CDDP or saline
solution (control group) presented obvious signs of physical
deterioration such as piloerection, ulcers, fallen hind legs and
bending. All treatments in general were tolerable maintaining a constant
increase in body weight during the whole experiment (Figure
5C ). However, the peptide and the combined treatment significantly
decreased the presence of physical deterioration signs and the
percentage of affected animals in comparison to CDDP administration
(Table 2 ).
In line with the reduction observed for tumor volume in this model and
the absence of physical deterioration signs, mice treated with the
peptide CIGB-552 and the combination improved life quality and increased
survival rate respect to CDDP or saline solution groups. The survival
rate for all treatments was significantly different compared to PBS
administration (p < 0,01) and the combination shows a tendency
to a superior overall survival in comparison to individual treatments,
marked by the fact that only the combined treatment group still have
live animals at the end of the experiment (Figure 5D ). The
treatment with the combination was very effective in comparison to
monotherapies, with a treated/control ratio (T/C) less than 15 (1.9), a
tumor growth delay (TGD) of 9.5 days respect to control group and
achieving a 98 % of tumor growth inhibition (Table 3 ). Thus,
our results demonstrate that CIGB-552/CDDP combination scheme elicits
its antitumor activity in vivo , with high tolerability and
effectiveness.
DISCUSSION
The generation of new therapeutic strategies and alternative treatments
has been a focus on cancer research. Drug combination in particular, is
getting attention as an interesting approach with a great current impact
in cancer therapy. The idea is to achieve a synergic or additive effect
between the drugs in the combination, in order to potentiate their
individual properties, reducing the doses but maintaining the
pharmacological effect, which allows the reduction of tumour growth and
metastatic potential, decreasing stem cell populations and inducing
apoptosis, and at the same time reducing toxicity and MDR (Tallarida
2001, Li, Zhao et al. 2014, Bayat Mokhtari, Homayouni et al. 2017,
Achkar, Abdulrahman et al. 2018).
In this work, we evaluated the pharmacological effects of the
combination between our anticancer peptide CIGB-552 and classic
antineoplastic agents currently employed in the clinics for lung cancer
treatment, such as CDDP and Paclitaxel. We evaluated potential
pharmacological interactions between CIGB-552 and both CDDP and
Paclitaxel through an in vitro drug combination assay in the
NSCLC cell line NCI-H460. We used two different combination schemes:
concomitant (both drugs acting at the same time) and pre-treatment
(preincubation with CIGB-552 and then add the other drug) similar to
clinical schemes used for cancer patients.
This drug interaction study showed a clear synergic effect between
CIGB-552 and both chemotherapeutic agents but the synergism and the
antiproliferative capacity were higher with CDDP compared to Paclitaxel
under the two treatment settings, and particularly under concomitant
conditions, where the synergism surface and the fraction affected were
greater. This indicate that co-administration of both drugs is essential
to obtain a better synergistic effect and a greater inhibition of cell
proliferation in non-small cell lung cancer lines. This study also
revealed some additivity between CIGB-552 and CDDP at middle
concentrations, which could potentiate their overall effect, although
synergism was the predominant interaction observed. Antagonism was only
present at higher concentrations in the concomitant condition,
suggesting that these drugs combined are more effective at middle and
lower concentrations. According to this, CDDP/CIGB-552 combination also
showed a best DRI at the lower concentrations, what also suggest that
CIGB-552 could help to reduce the doses of CDDP currently used in the
clinic, improving the patient responses to this antineoplastic agent.
Based on this result, we selected the combination of CIGB-552 and CDDP,
under concomitant scheme, to further evaluate its antitumor properties
and the synergism between both drugs.
Next, we investigated the capacity of the combination to modulate cell
cycle progression and induce apoptosis in NCI-H460 cells. Our results
demonstrated cell cycle arrest at G2/M phase and DNA fragmentation which
is an indicative of cell death. On the other hand, we confirmed
apoptosis induction in NCI-H460 cells in response to our products, which
was increased by the combination. Cell death by apoptosis is one of the
most important mechanisms that intrinsically controls malignant
transformation. Thus, apoptosis induction in tumor cells is considered a
key indicator of antitumor activity for new products/drugs and is also a
desirable effect for drug combinations (Meng, Wang et al. 2015). It has
been described that some chemotherapeutic agents such as CDDP, Topotecan
and Gemcitabine are able to induce apoptosis in NCI-H460 cells and other
NSCLC cell lines by a Caspase 8-dependent but death receptors and
Caspase 9-independent pathway, with mitochondrial permeabilization and
cytochrome c release as primary events (Ferreira, Span et al.
2000). In our study, the cleavage of PARP and Caspases 3, 8 and 9
confirmed apoptosis induction and suggested the activation of intrinsic
and extrinsic pathways by both products but mainly by the combined
treatment. Some authors have also showed that Cisplatin-acquired
resistance in other types of tumor like malignant pleural mesothelioma
is associated with a reduction in Caspase 8 activation and therefore
apoptosis induced by CDDP depends mainly on Caspase 9 activity (Janson,
Johansson et al. 2010) Our results demonstrated a preferential cleavage
of Caspase 9 in cells treated with the combination, particularly at 12
hours of incubation, thus the action of CIGB-552 could help to overcome
or decrease Cisplatin resistance in treated cells. Finally, we
corroborate apoptosis induction in NCI-H460 cells by Annexin V/PI double
staining, which revealed also a major percentage of apoptotic cells in
response to the combined treatment in comparison to the individual
drugs, confirming the synergic interaction between them.
Different authors have reported that the transcription factor NF-kB
interferes with the mechanism of action of antineoplastic drugs by
induction of antiapoptotic genes. Thus, the use of NF-kB inhibitors or
new drugs that target this molecular factor as adjuvant treatments,
could help to improve chemotherapy (Morotti, Cilloni et al. 2006,
Lagadec, Griessinger et al. 2008, Achkar, Abdulrahman et al. 2018).
NF-kB activation has been detected in many types of cancer including
small and non-small cell lung cancer and high expression of this nuclear
factor is correlated with progressive cancer and poor prognosis (Chen,
Li et al. 2011). NF-kB is induced in cancer cells in response to
chemotherapeutic agents like CDDP, as a tumor escape mechanism, related
with chemoresistance and insensitivity to chemotherapy (Galluzzi,
Senovilla et al. 2012, Godwin, Baird et al. 2013). Therefore, there are
many studies that demonstrate a synergistic activity combining an NF-kB
inhibitor with antineoplastic drugs. For example, Wang et al.
demonstrated that Gambogic acid (GA), a strong NF-kB inhibitor,
synergically potentiates CDDP-induced apoptosis in NCI-H460 cells (Wang,
Li et al. 2014). Gambogic acid has
antineoplastic and antiangiogenic properties and is currently in phase
II of clinical trials for NSCLC treatment (Wang, Deng et al. 2013).
Likewise, Bortezomib, a proteasomal inhibitor that decreases NF-kB
activation, enhanced the sensitization of bladder and cervical cancers
to CDDP (Miyamoto, Nakagawa et al. 2013, Konac, Varol et al. 2015). More
recently, the natural bioflavonoid Galangin (GG), which inhibits NF-kB
activity through downregulation of p-STAT3 signaling pathway, has
demonstrated to inhibit proliferation and enhance the apoptosis induced
by CDDP in human resistant lung cancer cells (Yu, Gong et al. 2018).
According to this, we corroborate synergism in antiproliferative effect
and apoptosis induction between CIGB-552 and CDDP in NCI-H460 cells. The
molecular mechanism of CIGB-552 is based on the inhibition of NF-kB
signaling pathway mediated by the stabilization and accumulation of the
intracellular protein COMMD1. (Fernández Massó, Oliva Argüelles et al.
2013). Thus, this could be a mechanism that plays an important role in
the synergic effects between both drugs and could contribute to decrease
cisplatin resistance in NSCLC. In addition, COMMD1 has demonstrated
strong anticancer and antimetastatic effects in different cancer models
(Van De Sluis, Mao et al. 2010, Riera‐Romo 2018). Furthermore,
Fedoseienko et al. demonstrated that nuclear expression of COMMD1
sensitizes tumor cells derived from advanced ovarian cancer patients to
platinum-based therapy. They suggest that COMMD1 modulate the G2/M
checkpoint, controlling expression of genes involved in DNA repair and
apoptosis (Fedoseienko, Wieringa et al. 2016). Then, is reasonable to
think that COMMD1 is also playing a key role in the molecular mechanism
that mediates CIGB-552 synergism with CDDP in NCI-H460 cells.
On the other hand, tumor cells have increased levels of ROS due to their
own metabolism deregulations, and it contributes to tumor development
and drug resistance. In line with this, the pharmacological manipulation
of the redox status to sensitize cancer cells to chemotherapeutic agents
is another attractive strategy to increase efficacy and avoid MDR.
(Dayem, Choi et al. 2010, Ma, Yang et al. 2014). In this work, we
demonstrated that the combination of CIGB-552 and CDDP increases
intracellular levels of ROS at short or prolonged exposure in NCI-H460
cells and do not have effect on normal cells from the same localization,
like MRC5 cells. These results suggest that selective induction of
oxidative stress could be an additional mechanism by which CIGB-552/CDDP
combination elicits its antitumor effects in NSCLC; an important
advantage of the combination compared to CDDP monotherapy.
One of the mechanisms underlying CIGB-552 cytotoxicity in NCI-H460 cells
is the COMMD1-mediated inhibition of SOD1 enzyme and the subsequent
induction of oxidative stress (Fernández Massó, Oliva Argüelles et al.
2013). CDDP also cause an unspecific production of high ROS levels,
which constitutes one of the main reasons of its toxicity (Chirino and
Pedraza-Chaverri 2009). Therefore, the combination with CIGB-552 could
contribute to reduce the nephrotoxicity and lymphopenia induced by CDDP
in cancer patients.
Our results showed that CGB-552 induces oxidative stress as an early
event, probably by COMMD1 stabilization and COMMD1-dependent SOD1
inhibition, whereas CDDP triggers ROS accumulation later in time, as a
secondary event, derived from its sequential enzymatic
biotransformation. This suggests that the peptide specifically
sensitizes tumor cells to CDDP through the modulation of the redox state
and consequently, both drugs combined generate sustained oxidative
stress that reinforces apoptosis induction and achieves a higher
antiproliferative effect (Figure 6 ). Besides, both products are
modulating the same cellular process but acting trough different
pathways, which could also explain the synergic interaction between
them. This idea has been supported by other authors, who point out
oxidative stress modulation as an interesting strategy to eliminate
cancer cells and sensitize them to chemotherapeutic treatments
(Trachootham, Alexandre et al. 2009, Raj, Ide et al. 2011, Gorrini,
Harris et al. 2013). For example, small molecules such as Resveratrol
(trans-3, 4′, 5-trihydroxystilbene) and Phenothiazines are able to
sensitize human colon and lung cancer cells, respectively, to the action
of different chemotherapeutic agents such as 5-Fluoracil (5-FU) to colon
cancer and Bleomycin and CDDP to lung cancer, through the modulation of
intracellular oxidative stress response (Santandreu, Valle et al. 2011,
Zong, Hååg et al. 2011). Another example is the natural compound
Shikonin, which triggering
intracellular oxidative stress in colon cancer cells but not in normal
cells, potentiated CDDP-induced DNA damage, followed by increased
activation of the mitochondrial apoptotic pathway. The use of
antioxidants and ROS scavengers revealed that ROS are essential to the
synergism observed (He, He et al. 2016).
The results obtained in vitro were corroborated in vivo in
a mouse model of TC-1 lung cancer, a recognized animal model for NSCLC
(Tanaka, Delong et al. 2005). Co-administration of CIGB-552 and CDDP
lead to a significant inhibition of tumor growth, with increased overall
survival in treated animals and increasing the life of quality in
comparison to control mice or mice treated with the drugs separately. In
addition, the combination with CIGB-552 was able to significantly
decreases the signs of physical deterioration associated with CDDP
administration. This confirms that both drugs are acting synergically to
achieve a better antitumor response and is correlated with the behaviour
observed in in vitro studies. Similar results were obtained by
Wang et al. with GA, which sensitizes human lung cancer cells to CDDPin vitro , by NF-kB inhibition and ROS intracellular accumulation,
and was also effective in vivo , in a A549 xenograft mice model,
where the combined administration with CDDP significantly decreased
tumor volumes of treated animals, without body weight loss or associated
toxicity (Wang, Li et al. 2014). In the same way, Shikonin that
eliminates human colon cancer cells and sensitizes them to CDDP-induced
apoptosis through the selective induction of oxidative stress, was also
able to inhibit tumor growth in a HCT116 xenograft model in nude mice
(He, He et al. 2016). More recently, Hsu et al. demonstrated the high
potential of another natural compound, Withaferin A (WA) in lung cancerin vitro and in vivo . As occurred with CIGB-552, WA is
selectively cytotoxic to different human lung cancer cells including
various NSCLC cell lines, inducing apoptosis and increasing the
intracellular accumulation of ROS as its antitumor mechanism. In
addition, it decreases lung tumorigenesis in vivo in a NSCLC
model of H441-L2G bioluminescent cells implanted in nude mice. Similar
to our results, WA and CDDP synergically inhibited NSCLC cell
proliferation in a drug combination assay and WA enhanced CDDP
cytotoxicity and antitumor activity in cell cultures and tumor spheroids
(Hsu, Chang et al. 2019).
Taken together, all these findings demonstrate that targeting NF-kB
activity and ROS response in tumor cells is an effective therapeutic
strategy in NSCLC and other types of cancer, which can improve the
response to different chemotherapeutic agents but particularly to CDDP,
achieving synergistic effects and decreasing CDDP resistance. The
combination of CIGB-552 and CDDP is able to modulate both molecular
pathways, representing an important advantage in NSCLC treatment. Based
on the presented evidence we propose a model in which CIGB-552
sensitizes lung cancer cells to CDDP through ROS accumulation and NF-kB
inhibition, achieving synergism in apoptosis induction and reduction of
tumor growth. This research is the first preclinical evidence about the
combination of CIGB-552 and CDDP in the context of NSCLC and gives
important findings that support the use of CIGB-552 as an adjuvant
treatment in the clinics.
CONCLUSIONS
CIGB-552 is a new cancer targeted therapy that acts synergically with
CDDP to inhibit proliferation and tumor growth of NSCLC in vitroand in vivo . The combination of CIGB-552 with chemotherapeutic
agents like CDDP is an attractive strategy to selectively induce ROS and
apoptosis in lung cancer cells, improving the antitumor efficacy of CDDP
but decreasing its associated toxicity. This work also shows scientific
evidence that could help in the rational design of a combined treatment
for lung cancer based on CIGB-552, to be tested in clinical trials.
ACKNOWLEDGMENTS
We would like to thank Yasser Perera Negrin, Isbel Garcia Figueredo and
Monica Bequet Romero for their advices and collaboration on this
experimental work.
CONFLICT OF INTERESTS
Authors do not declare any conflict of interests associated to this
work.
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TABLES
Table 1. Combination parameters under concomitant treatment for
maximal, minimal and median concentrations of CIGB-552, CDDP and
Paclitaxel in NCI-H460 cells.