Synergic Effects of Anticancer Peptide CIGB-552 and Cisplatin in Lung Cancer Models


 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 demonstrated in 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.


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. 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 rst-line treatment and 6 cycles of docetaxel (or equivalent taxol drug) as a second-line regimen. Both regimens employ unspeci c 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 longterm application in cancer therapy in addition to other negative impacts such as multidrug resistance (MDR), mutagenicity and teratogenicity (Bordoni 2008 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 rst 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 classi ed 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 bene t of the best combination and therapeutic scheme was corroborated in vivo in a mouse model of lung cancer.

Reagents and Chemicals
All regents and chemical substances used in this study were purchase from Sigma-Aldrich. Culture media and cell culture material were obtained from Life Technologies (USA), GE Healthcare, and Greiner. Fetal bovine serum (FBS) was acquired from HyClone, Logan, UT. 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 puri ed by reverse-phase-HPLC to > 95% purity on an acetonitrile/H 2 O trifuoracetic acid gradient and con rmed 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 vitro and in vivo.

Cell lines
NCI-H460 (human non-small cell lung carcinoma), MRC-5 cells (human embryonic lung broblasts) 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% CO 2 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 4x10 4 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 tree times 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 (4x10 4 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 xed 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 tting the DNA distribution to a diploid DNA content for cell cycle pro ling, cellular debris and doublets were properly excluded by gating out in FL3-A vs. FL3-W two-parameter dot plots. analyzed according to the western blotting technique previously described (Burnette 1981). Brie y, 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 speci c bands were visualized using chemiluminescence reagents followed by exposure to standard X-ray lms.
2.9. 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 (1x10 5 cells 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 6x10 3 and 1x10 4 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 (H 2 O 2 ) 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 uorescence 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 uorescence intensity as fold of control for all treatments. We processed images from three independent experiments.
To con rm the induction of oxidative stress in NCI-H460 cells, we perform a ow cytometry-based assay for HE detection in treated cells according to the method described by Walrand and colleagues (Walrand, Valeix et al. 2003). Brie y, we seeded 1 x 10 6 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 (H 2 O 2 ) 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 2x10 4 events were analyzed and HE positive cells were quanti ed. 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 5x10 4 cells per animal of the murine tumor cell line TC-1 on the right ank, according to the procedures reported for this animal model (Tanaka, Delong et al. 2005). When tumors reached 70-90 mm 3 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 rst 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 rst 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 width 2 /2. Survival rate was daily registered during the experiment and only when tumor volumes reached 2,000 mm 3 , the animals were sacri ced 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 uorescence 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 ow 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 signi cance of differences in survival rates was determined by logrank test p * <0,05. All statistical analysis were done using the software GraphPad Prism 7.

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 IC 50 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 IC 50 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 ( Fig. 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 signi cance 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  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 bene cial 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 e cacy 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 IC 50 of CIGB-552 (37.5 µM) and above the IC 50 of CDDP (5 µM). According to this, we decided to evaluate the effect of both drugs on subsequent experiments at these particular concentrations. 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 con rm apoptosis induction in NCI-H460 cells by the Annexin V/Propidium iodide (AV/PI) double staining, evaluated by ow 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 (Fig. 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 (Fig. 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 con rming 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 (H 2 O 2 ) was used as positive control for the experiment. Both, the treatment with CIGB-552 and the combination promoted the increase of uorescence in treated cells, demonstrating the accumulation of O2•−, in contrast to CDDP that showed a minor effect (Fig. 4A).
Comparing the mean uorescence 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 (Fig. 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 broblasts (MRC-5 cell line). As we expected the evaluated products do not stimulate O2•− accumulation in these cells (Fig. 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 ow cytometry. We used H 2 O 2 (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 uorescence obtained after 1 hour of exposure, we con rm 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 signi cant increase in the percentage of HE positive cells (13.8%), demonstrating synergy in the induction of oxidative stress mediated by the products (Fig.  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-speci c toxicity promoted by Cisplatin, keeping the increase of ROS as a selective mechanism of cytotoxicity in tumor cells.  Fig. 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 e cacy in colon cancer models to select the dose and administration route, following the experimental design described in materials and methods (Sect. 2).
The administration of CIGB-552 and CDDP on tumor-bearing mice led to a signi cant reduction of tumor growth compared with the group treated with saline solution (p < 0,05) ( Fig. 5A and B). In addition, mice treated with the combination exhibited signi cantly 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 (Fig. 5C). However, the peptide and the combined treatment signi cantly 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 signi cantly 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 (Fig. 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, 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 nonsmall 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 con rmed 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 con rmed 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 Cisplatinacquired 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 demonstrated that Gambogic acid (GA), a strong NF-kB inhibitor, synergically potentiates CDDP-induced apoptosis in NCI-H460 cells . 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 bio avonoid 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 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 speci cally 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 (Fig. 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 ( 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 signi cant 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 signi cantly decreases the signs of physical deterioration associated with CDDP administration. This con rms 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 CDDP in 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 signi cantly decreased tumor volumes of treated animals, without body weight loss or associated toxicity

Conclusions
CIGB-552 is a new cancer targeted therapy that acts synergically with CDDP to inhibit proliferation and tumor growth of NSCLC in vitro and 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 e cacy of CDDP but decreasing its associated toxicity. This work also shows scienti c 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.

Declarations
Funding: There is no funding source associated with this work Con icts of interest/Competing interests: Authors do not declare any con ict of interests associated to this work.
Availability of data and material: We declare total transparency with the data generated and presented in the above manuscript.
Code availability: Not applicable.
Authors' contributions: Yolanda, Brizaida, Maribel, Julio, Cossio,Hilda: Substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work. Consent to participate: Non applicable.

Consent for publication:
Non applicable. We are using our own experiments and data.