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 (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 (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 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 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 cancer in 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.