From the molecular and physiological points of view, the set of pathological conditions commonly described as cancer presents a complex phenomenology that influences the design of therapeutic strategies. In particular, the use of anti-cancer pharmacological therapies directed at specific molecular targets is usually considered among the best strategies to alleviate the disease. However, due to the dense and robust network of (abnormal) interactions set-up by the biomolecular and physiological alterations typical of cancer, the efficacy of such targeted therapies is often challenged (de Anda-Jáuregui et al., 2015). In such cases is necessary to resort to less-specific drugs (chemotherapeutics), whose mechanisms of action are mostly cytotoxic. Cytotoxic therapy presents a number of disadvantages when compared with its targeted counterparts. Most cytotoxic compounds are highly unspecific and damaging at the organismal level. For instance, metal-based chemotherapy (such as the alkylating agent cisplatin) presents DNA damaging activity and induces cell death (primarily by apoptosis but also by necrosis) in an uncontrolled way (cisplatin is a cell-cycle unspecific drug). Long term effects in humans include anemia and leukopenia, liver and kidney damage, neurological diseases (such as peripheral neuropathy), serum electrolyte disturbances and vascular toxicity among others.

Research in chemotherapy aims at developing alternative cytotoxic agents to be used as anti-cancer drugs. Such drugs will ideally avoid some of the most harmful side effects of current metal-based chemotherapy. In this regard, copper-based drugs known as Casiopeínas present a series of advantages, such as the fact that copper-compounds may be metabolized, thus reducing indirect toxicity and metal-related poisoning (Ruiz-Azuara, 1997; Bravo-Gómez et al., 2009; Valencia-Cruz et al., 2013). Aside from this, the pharmacological dosage is lower than that of competing therapies.

To evaluate the systemic effect of such novel cytotoxic compounds, an integrative view is desirable since—as we already mentioned—secondary immediate and long-term effects arise from their use. Systems biology provides precisely such view by incorporating elements from high-throughput omic technologies with strong computational and analytical tools. In the past, some systems biology attempts to study the effects of Casiopeínas have been carried out (Gutiérrez et al., 2013; Valencia-Cruz et al., 2013). Those efforts, however, were focused in the anti-proliferative effects (increasing cell death and arresting cell cycle) which are of course central to understand the main mechanisms of antineoplastic action. Other biological processes may, however, be affected by Casiopeínas, so that new molecular functions—that may add to the therapeutic efficacy and possibly to collateral effects—are expected to rise. It is desirable then, to account for these effects in a quantitative/qualitative manner aiming at a mechanistic characterization (Hernández-Lemus, 2013).

Genome-wide gene expression analysis (GEA) provides an appropriate tool to study systems-level biomolecular processes responsible for phenotypic traits. However, GEA alone falls short represent all the complex interactions present among such processes. For this reason, Pathway Analysis methods have been developed to improve the predictive power derived from such high-throughput experiments (Tarca et al., 2009; Vaske et al., 2010; Drier et al., 2013; Krämer et al., 2013; Wang et al., 2013; Huang et al., 2014; Verhaegh et al., 2014). Pathway level studies have revealed new insight that may lead to a functional picture of complex phenotypes such as the ones associated with cancer (Lohr et al., 2012). Such methods are built upon system-level analytics, consisting in probabilistic and computational modeling and simulation strategies. Those strategies are performed in large amounts of experimental data contained in carefully curated information repositories, that may be in the form of structured Knowledge-based data bases, but also in unstructured data-mining and literature-search applications.

To date, a vast majority of the pathway analysis tools is based on statistical tests on the number of differentially expressed genes that are enlisted as part of some biological pathway. These methods are commonly referred to as enrichment or over-representation methods. These approaches, though very simple, have provided an additional dimension (aimed at a functional description) to the large scale molecular study of biological systems. The analysis of cellular processes under the perspective of biological interaction networks has revealed foundational principles; these in turn may lead to the formulation of functional and mechanistic hypotheses to be further tested and refined. In this regard, knowledge-based sources such as Ingenuity Knowledge-Base (IKB, Krämer et al., 2013) present an effective alternative (based on highly curated information sources, relying on detailed experimental evidence and extensive literature references) to large scale probabilistic and computational modeling, especially useful when facing small sample counts, as is the case here.

Cancer is a complex disease, with a plethora of intricate mechanisms that determine the features of specific phenotypes which in turn give place to distinct prognosis and treatment. Chemotherapy is one of the most used treatments to face this condition. Casiopeínas have shown an important chemotherapeutic effect on cancer cell lines (Valencia-Cruz et al., 2013). The intrinsic route to apoptosis is favored by caspase-3 activation via cyt C overexpression, high ROS concentrations, as well as cell cycle arrest; whereas the extrinsic route to apoptosis is somehow diminished due to very low expression of procaspase-8 and caspase-8. Antiapoptotic processes are switched-down, due to low levels of Bcl-2. Furthermore, copper-based therapy could be more convenient than other cytotoxic agents in terms of a decrease in side effects, since copper, being an essential trace element can be metabolized. Casiopeínas reach the membrane of mitochondria and exerts a pro-apoptotic effect (Valencia-Cruz et al., 2013), as can be seen in Figure 3. As it was previously mentioned, Cas II-gly activates molecules that participate in oxidative stress processes and reduces cell viability by the intrinsic apoptotic pathway. In this work, we developed an integrative systems biology approach to elucidate the effect of Cas II-gly treatment in HeLa cell lines, observing new features, namely, inhibition of G1/S transition phase, decrease of cell transformation process and a decrease of cell migration.

Cas II-gly promotes apoptosis and metal ion signaling responses (Valencia-Cruz et al., 2013). At the same time, with the results shown by this analysis, we demonstrate that Cas II-gly inhibits cell proliferation, cell migration, transformation of cells as well as cell cycle progression in HeLa cell lines. Furthermore, this treatment promotes the liver repairing system as well as the activation of molecules related to decrease of liver damage.

Regarding the inhibitory effect that Cas II-gly has on cell cycle and transcription, it can be observed in Figures 5–7, that the inducer molecules involved in those processes are underexpressed. A good example of this is depicted in Figure 5, where a considerable subset of the molecules involved in the estrogen-mediated G1/S phase progression are underexpressed. A similar effect is observed in Figure 6, where the upstream molecules FOXM1 and NTRK2 are downregulated. This downregulation event affects the cell-division molecules CDC25A, CCND1, CCNE1, MYC, SKP2, and CCNA2. Interestingly, FOXM1 underexpression abolishes the negative feedback with CDKN1A (Wang et al., 2002, 2007); this cyclin-dependent kinase inhibits the S-phase entry of fibroblasts (Sherr and Roberts, 1999). It is worth to mention that, despite the fact that the network depicted in Figure 6 corresponds to processes diminished in fibroblasts and bone cancer cell lines, the molecules involved in these processes are ubiquitous in all cell types. Furthermore, some of these molecules are known to be markers of tumor growth as well as cell cycle progression. As it was explained before, Ingenuity Pathway Analysis contains a large database of interactions. Those interactions have been highly curated by literature or ex professo experiments. The observed processes of Figure 6 correspond to interactions of those molecules observed on those cell types. However, the interactions are also common in many other cell types. Based on that, to extrapolate the effects that Cas-II gly exerts on fibroblasts to HeLa cell lines may be appropriate in terms of the set of molecules implied in those processes.

By observing Figure 7, it can be noticed that MED1 exerts a repression on CDKN1A (p21) which in turn inhibits cell migration (Lee et al., 2013); the same is true of HMOX1 (Lu et al., 2012); thus, concomitant overexpression of these molecules exert a negative role in migration. Other molecules involved in the process of cell migration are underexpressed; all of those molecules have a pro-migration role. Hence, their underexpression generates a global anti-migration effect. This could be relevant in terms of the complementary role that Casiopeínas may have as a therapeutic treatment: Casiopeínas act as a cytotoxic (Bravo-Gómez et al., 2009; Gutiérrez et al., 2013; Valencia-Cruz et al., 2013) and concomitantly they may exert an inhibitory effect on cell migration.

In this work, we have performed pathway analysis with two different but complementary strategies. The general mechanism of action of Cas II-gly is known in terms of the damage to DNA and mitochondrial stress. However, the whole set of different effects that this drug exerts on the cell is not well-known. With the analysis performed here, we observe that some molecules that have been related to liver damage and repairing are differentially expressed in the treated cells respecting the control ones. These molecules are ubiquitous in any cell type: JUND, IL6, HMOX1, ADM, MAPK14, DDT3. The importance of these results lies on the fact that these molecules are consistently associated with protection from hepatic damage (at the systemic level). The experiment shown here, was performed in a cervical cancer cell line. However, the effect of Cas II-gly in this cell line activates (or deactivates) specific molecules that have been observed to perform a particular effect at the systemic level. It remains to be tested (by means of in vitro but even more importantly, by in vivo models and clinical experiments) whether the molecular pathways activated by Cas II-gly are actually able to induce such liver protecting processes in living subjects.

Cas II-gly is a potent and promising chemotherapy. In this regard, the results presented here corroborate the potential usage of this drug as an anticancer therapy. One of the most relevant results is related to liver damage. In Figure 8 can be observed that almost every molecule in the figure is overexpressed, and these molecules inhibit liver damage. Activation of molecules downstream of MAP2K3 allows a protection of the liver, with the exception of p21 (CDKN1A). The overall effect of those interactions is hepatoprotective. The last result might be helpful in terms of the clinical potential, given the fact that the majority of metal ion therapy is based on platinum, and it is also well-known that platinum cannot be naturally metabolized. Furthermore, the GO (biological processes) terms in the overexpressed genes sets are enriched for the response of the metal ion signaling pathway, reflecting the fact that cells are facing to this external agent.

Further steps toward a complete understanding of the mechanisms of action of Casiopeínas, involve a comparative analysis in vivo. Experiments in this regard are currently on their way. Such analyses, as well as a toxicological study (phase I clinical trial) in human subjects are ongoing within our group. In the meantime, the systems biology approach presented here, shows general and also specific features of the regulatory effect that Cas II-gly may exert, and this knowledge could be relevant to provide a more integrated overview of the processes in which novel anticancer therapies are involved, looking for the understanding of global chemotherapeutic mechanisms.

JE performed analysis and calculations, contributed with algorithms and analytical methods, collaborated in the discussion, collaborated in writing the manuscript, EH conceived and designed the study, contributed with algorithms and analytical methods, performed analysis and calculations, collaborated in the discussion, collaborated in writing the manuscript. CM performed the experiments, collaborated in the discussion, collaborated in writing the manuscript. LR synthesized the drugs, collaborated in the discussion, collaborated in writing the manuscript. All authors read and approved the final manuscript.

This work was supported by CONACYT (grant no. 179431/2012), as well as by federal funding from the National Institute of Genomic Medicine (Mexico). Additional support has been granted by the National Laboratory of Complexity Sciences (grant no. 232647/2014 CONACYT).