Non-Platinum Metal Complexes as Potential Anti-Triple Negative Breast Cancer Agents

Breast cancer (BC) is the most common cancer in women worldwide, with a mortality rate that has been forecasted to rise in the next decade. This is especially worrying for people with triple-negative BC (TNBC), because of its unresponsiveness to current therapies. Different drugs to treat TNBC have been assessed, and, although platinum chemotherapy drugs seem to offer some hope, their drawbacks have motivated extensive investigations into alternative metal-based BC therapies. This paper aims to: (i) describe the preliminary in vitro and in vivo anticancer properties of non-platinum metal-based complexes (NPMBC) against TNBC; and (ii) analyze the likely molecular targets involved in their anticancer activity.


Introduction
Breast cancer (BC) is, despite therapeutic advances, the most common cancer among females (1.5 million women worldwide each year) and the leading cause of death from cancer in women (570,000 in 2015).Furthermore, its incidence is expected to rise in the coming decades [1].This global increase in the BC burden is mainly attributed to exogenous factors such as reproductive and lifestyle factors; however, genetic risk factors such as mutations in breast cancer susceptibility gene 1 (BRCA1) and gene 2 (BRCA2), which are associated to tumors with more aggressive phenotypes, play an important role in the etiology of this type of cancer [2,3].
BC is classified according to the expression of immunohistochemical markers in luminal A, luminal B, HER-2 positive and triple negative subtypes, being the triple negative subtype further divided into A (luminal-like) and B (basal-like) [4,5] (Table 1).The basal-like cancers account for 60-90% of triple negative cases, with the BC subtype having the worst prognosis.Compared with the other subtypes, triple negative breast cancer (TNBC) is more likely to be diagnosed at a younger age; it is more frequently associated with mutations in the BRCA; it usually has rapid growth and large size; it frequently overexpresses genes involved in drug-desensitizing mechanisms (for example, ABCC1 or MRP1 (multidrug resistance protein 1)); and it has a high rate of early recurrence and of distant metastasis (brain, lung, bone, and liver), and a low disease-specific survival [6].Furthermore, due to the lack of specific molecular targets in this subtype of BC [7], which makes its treatment difficult, chemotherapy remains an essential component for the management of TNBC, both adjuvant and neoadjuvant therapy [8,9].
This paper aims to: (i) describe the preliminary in vitro and in vivo anticancer properties of non-platinum MBC (NPMBC) against TNBC; and (ii) analyze the likely molecular targets involved in their anticancer activity.

Anticancer Effects of Non-Platinum MBC (NPMBC)
The mechanisms behind the anticancer effects of NPMBC are not fully understood, but known responses to them include the following: (i) suppression of cancer cell viability (Table 2) in association with induction of cell death (Tables 3 and 4); and (ii) inhibition of metastatic processes (Table 5).Furthermore, a study suggests that NPMBC could also inhibit angiogenesis [55]; however, because of scarce evidence, no definitive conclusion can be drawn on the role of these compounds in the regulation of blood vessel formation.In any case, it is thought that NPMBC are frequently multi-targeted and attack different biochemical pathways simultaneously [53,66,68], sometimes in a synergic way [37,62].Cellular studies constitute an important step in the development of drugs; however, demonstrating efficacy in suitable animal models and validating cellular observations in vivo are a condition sine qua non for clinical trials investigating potential anticancer agents.Inconsistencies in results between in vitro and in vivo systems concerning the anticancer effect of NPMBC can be related to differences in their pharmacokinetic behavior, thus their study is necessary.In this way, NPMBC seem to have good in vivo pharmacokinetic properties.They are absorbed quickly into plasma [16,39,44,52], the circulation time in the bloodstream is long [42], their uptake in the tumor tissue is significant [16,28,32,37,39,41,42,44,46,51,52,60,70], their accumulation in organs (brain, liver, kidney, spleen, heart, lung, and intestine) is low [16,28,39,41,42,44,51,52,55] and the clearing rate is good [51].
A large proportion of current knowledge about the therapeutic potential of NPMBC is derived from in vitro and in vivo studies performed using TNBC cell lines.TNBC MDA-MB-231 cell models are considered the most reliable models of TNBC [71,72], so this cell line is the world's most commonly used human TNBC cell line.However, this raises the issue on how representative this cell line is of the broad spectrum of TNBCs (Table 1).

Suppression of Cancer Cell Viability in Association with the Induction of Cell Death
NPMBC-mediated inhibition of breast carcinogenesis is associated with reduced cellular proliferation (Table 2) together with the induction of programmed cell death (Tables 3 and 4).Apoptosis is by far the primary mode of programmed cell death (Table 3), but sometimes it is coupled to the induction of autophagy, as can be suspected from the few studies which have been carried out (Table 4).
NPMBC display good cytotoxic behavior in vitro and are able to reduce the growth of primary tumors (Table 2).NPMBC inhibit the growth of TNBC cells better than non-TNBC cells [66].Furthermore, NPMBC of which effects are dose- [49,53,55,67] and time- [63,67] dependent could be more cytotoxic than platinum-based drugs [52,53,57,58,68].For example, Biancalana et al. [57] observed that ruthenium complexes have good cytotoxic activity, with IC 50 values substantially lower than the values obtained with cisplatin on MDA-MB-231 cells.These differences could be due to the fact that they have different mechanisms of action; for example, the activation of p53-dependent or p53-independent checkpoints by cisplatin or ruthenium complexes, respectively [9,52,54,68].Since platinum-based drugs and NPMBC act on different pathways, NPMBC could be a new therapeutic option for patients with TNBC resistant to platinum-based drugs.
Avoiding apoptosis is a hallmark of cancer and an important mechanism in resistance to therapies.Apoptosis is preferred over necrosis because the latter elicits inflammation and undesirable immunogenic responses [73].NPMBC induce significant apoptotic activity in TNBC cells which can be mediated by multiple mechanisms (Table 3).When MDA-MB-231 and BT-20 cells were treated with 25 µM copper salicylate phenanthroline complexes, more than 80% of TNBC cells underwent apoptosis through down-regulation of the anti-apoptosis proteins Bcl-2, Bcl-xL and survivin and up-regulation of cleaved PARP [49].These findings confirm the participation of protease enzyme caspases in the programmed cell death induced by NPMBC.However, NPMBC can also activate caspase-independent pathways acting on mitochondria.For example, when MDA-MB-231 cells were treated with 10 µg/mL of silver nanoparticles, high intracellular levels of reactive oxygen species (ROS) were observed at 24 h of incubation [67].These free radicals destabilize the mitochondria and induce apoptosis [74] acting on Bcl-2 family proteins [75].Considering that TNBC is more susceptible to drugs that cause oxidative stress than other BC types [66], these compounds could be a hopeful alternative method for its treatment.Besides what was mentioned above, we must not forget that the efficacy of several NPMBC relies on their capacity to influence the tumor-host interaction and modify the immune microenvironment, inducing a form of apoptosis in cancer cells known as "immunogenic cell death".Thus, Montani et al. [55] observed that ruthenium complexes reverse tumor-associated immune suppression leading to the activation of an immune response specific for TNBC cells.
The NPMBC can be synthesized by chemical [76], physical [77] and biological methods [35], all with cytotoxic activity against TNBC cells.Although chemical and physical syntheses have been the most widely used strategies, there is growing interest in the biosynthesis of NPMBC because of its numerous advantages (for example, economical, safe, and inert) [78,79].
The NPMBC differ in shape [34], size [31][32][33]42], dose [30,36,66], synthesis procedure (see previous paragraph), composition [50,56], capping agent [33,80], particle surface charges [31] and structure [56,68,69].All of these features can affect their cellular uptake and therefore also their cytotoxic activity.In general, modified small nanoparticles with low surface charges (−15 mV) have better cellular internalization than large nanoparticles, however both extremely small (<25 nm) and large (>80 nm) nanoparticles displayed a low cellular uptake [31,33].On the other hand, it has been demonstrated that replacing ruthenium with iron gives compounds with high antiproliferative activity (IC 50 = 0.09 vs. >30 µM), the compounds with the shortest carbon chain linking the two cyclopentadienyl rings being the most active [56].Finally, another interesting finding is the superiority of nanorods over nanospheres for photothermal cancer therapy (PCT) using gold nanoparticles [34].Despite this evidence, it has not been possible to establish a linear correlation between cellular responses and the characteristics of NPMBC because there is wide interstudy variability (for example, in relation to cell lines, measurement methods, etc.).
NPMBC possess cytotoxic activity per se [39,66,67,69]; however, their main applications are related to their ability to act as radiosensitizers in cancer cells through the induction of G2/M phase cell cycle arrest [36,47,66], as agents for PCT applications [14,28,29,37,63] or as transmembrane carriers for the controlled release and targeted delivery of anticancer drugs increasing their cellular uptake [30,39,40], among others [47,64].Cancer cells in a tumor are very heterogeneous, that is, they differ in marker expression, morphology, proliferation capacity, growth stage, etc.This heterogeneity increases the need for combining therapeutic agents with different action mechanisms.Although few studies have examined the role of NPMBC for combination chemotherapy, recently the utility of iron oxide magnetic nanoparticles as multidrug codelivery system for synergistic chemotherapy has been demonstrated [63].
The effects of NPMBC on TNBC cells, as well as on cancer cells from other solid tumors, is facilitated by abnormalities of the tumor vasculature, such as hypervascularization, high production of vascular permeability factors, etc. [81].However, intrinsic properties of NPMBC are not less important.For example, nanoparticles are characterized by their high permeability [82] and retention effect [83] which results in potentiation of the cytotoxic effects with minimal side effects [39,49].When the tyrosine kinase inhibitor ZD6474 was conjugated with gold nanoparticles, besides inducing a higher antiproliferative effect than the ZD6474 alone under in vitro conditions, it also reduced the size of tumors induced in mice more efficiently than the drug alone [44].These properties could be useful to counteract drug resistance mechanisms, the main limitation to the success of conventional therapy.In this sense, Conde et al. [41] showed that gold nanoparticles loaded with 5-fluorouracil (IC 50 = ~0.5 nM) decrease the viability of 5-fluorouracil-resistant MDA-MB-231 cells by silencing the cell surface efflux pump MRP1.
One of the concerns for developing NPMBC as therapeutic agents for TNBC treatment could be their toxicity.However, several studies have proven their harmlessness in normal cells [39,49,52,57,66,84]. Ruthenium(II) paracyme complexes showed a moderate selectivity for TNBC cells, so normal cells (human skin fibroblasts) were less affected than MDA-MB-231 cells [57].Likewise, when copper salicylate phenanthroline complexes [49], ruthenium complexes [55] or gold nanoparticles [28,37,39] were administered to mice, they were well tolerated as evidenced by the lack of weight loss, behavior of the animal or examination of organs.
It is hypothesized that, because of their capacity for self-renewal, differentiation and apoptosis-resistance [85], cancer stem cells (CSCs, also referred to as tumor-initiating cells) could be responsible for the cancer formation [61].What is more, they could be resistant to conventional treatments and contribute to the recurrence after treatment [86].Gold nanoshells seem to sensitize breast CSCs to radiation therapy mediated by the activation of the heat shock proteins (HSP) HSP40, HSP60, HSP70 and HSP90α [45].Likewise, CSCs' ability to self-renew might be inhibited by iron oxide nanoparticle-mediated PCT [61] or iron oxide nanoparticles-linked leptin antagonist [64] which has been demonstrated through the reduction of mammospheres/tumorspheres of MDA-MB-231 and SUM 159 cells [61,64].Although these findings are hopeful, new strategies for improving the intratumoral distribution of NPMBC are necessary to achieve a homogeneous nanoparticle distribution within the tumor without depositing them in surrounding healthy tissue [70].

Migration, Invasion and/or Metastasis
Metastasis is a cascade of events where tumor cells disseminate from the primary tumor to distant sites, with the migration of tumor cells being a prerequisite for tumor-cell invasion and metastasis (Figure 1) [87].Thus, any factor which regulates this process might be a target for anti-metastatic therapeutic strategies (Table 5).The poor prognosis of the triple negative subtype reflects the aggressive biology of this disease and the difficulty of its treatment, especially when it metastasizes.
Study of the anti-metastatic effect of NPMBC on TNBC cells is very recent.It has been shown that NPMBC, besides enhancing the potential of anticancer therapies [43,44,62], can be used as monotherapy [43,51] to prevent cancer cell migration, invasion and/or metastasis.These findings were confirmed by Paholak et al. [61] using in vivo models.When immunocompromised NOD/SCID mice were inoculated with TNBC cells, it was observed that those mice that received nanoparticle-mediated PCT prior to surgical tumor resection had lower incidence of metastasis to the lung than those which had been treated only with surgery.If these findings were observed in humans, combination therapy consisting of nanoparticle-mediated PCT and surgery would provide a treatment option for patients with metastatic breast cancer, a disease which nowadays has no cure [88].
Unfortunately, little is known about the mechanisms underlying the anti-metastatic effect of NPMBC (Figure 1 and Table 5).However, available evidence suggests that their action could be mediated by the inhibition of matrix metalloproteinases (MMPs) [59], enzymes with the ability to degrade extracellular matrix proteins, after interaction with the αvβ3 integrin receptor [62].Likewise, it has been suggested that NPMBC could inhibit epithelial-mesenchymal transition [65] and modify the structure of the actin cytoskeleton, interfering with the function of integrins [58,59] and under-regulating the phosphorylation of the focal adhesion kinase (FAK) [51].No less important are the studies which highlight their role in inflammation [89,90].On the one hand, it has been observed that doxorubicin-hyaluronan conjugated iron oxide nanoparticles reduce the secretion of the pro-inflammatory IL-6, an important prognostic indicator in patients with TNCB [91], and which is thought to contribute to their invasiveness [92]; on the other hand, it has been suggested that iron oxide nanoparticle-mediated hyperthermia could trigger the release of inflammatory mediators which would induce a systemic cancer-specific immune response in which cytotoxic T-cells would recognize and inhibit distal cancer cells [89].
One of the major limitations of anti-metastatic chemotherapy, as has already been mentioned for the treatment of primary tumors, is the toxicity which underlies the importance of targeted therapies.Anticancer drugs should be delivered and act only at the site of action.In this respect, nanoparticles are worthy of mention because they more effectively inhibit the migration of TNBC cells compared to non-tumor cells [51].Sarkar et al. [44] found potentiation of the anti-migration and anti-invasion effect of the tyrosine kinase inhibitor ZD6474 when it was conjugated to gold nanoparticles synthesized in micellar networks of an amphiphilic block copolymer.On the other hand, the conjugation of interfering RNA (siRNA) to nanoparticles in order to suppress the expression of proteins is another interesting approach.It has been shown that chitosan-layered gold nanorods can be used for silencing genes related to the invasion of TNBC cells [43].Another strategy which enables the selectivity for tumor cells to be enhanced is by attaching nanoparticles to ligands that bind to receptors expressed on the surface of cancer cells, as has been highlighted by Ahir et al. [51].Considering that folate receptor expression is elevated in BC cells [93], this receptor could be a good target for cancer treatment.Copper oxide nanowires conjugated with folic acid prevent the motility of MDA-MB-231 cells, an effect that has been validated in vivo [51].and its inhibition by non-platinum metal-based complexes (NPMBC).Metastasis is a sequential and interrelated multi-step process that consists of four main essential steps: migration to adjacent tissue after detachment from a primary tumor; local invasion of cancer cells into adjacent tissue; transendothelial migration of cancer cells into vessels (intravasation); transport of cancer cells through the circulatory system with extravasation of them in a secondary organ (brain, lung, liver, bone).The antimetastatic effect of NPMBC has been associated with their ability: to inhibit matrix metalloproteinases and its inhibition by non-platinum metal-based complexes (NPMBC).Metastasis is a sequential and interrelated multi-step process that consists of four main essential steps: migration to adjacent tissue after detachment from a primary tumor; local invasion of cancer cells into adjacent tissue; trans-endothelial migration of cancer cells into vessels (intravasation); transport of cancer cells through the circulatory system with extravasation of them in a secondary organ (brain, lung, liver, bone).The anti-metastatic effect of NPMBC has been associated with their ability: to inhibit matrix metalloproteinases (a); to inhibit mesenchymal markers such as vimentin (b); to interfere with the function of integrins (c); and to modify the immune/inflammatory response (d,e).

Conclusions
Anticancer drugs with new molecular mechanisms of action are necessary for chemotherapy treatment of TNBC, with NPMBC emerging as an upcoming treatment modality.It has been suggested that NPMBC could have an effect on different TNBC cell lines, both in vitro and in vivo.In this way, it has been shown that NPMBC are responsible for activating programmed cell death and exhibiting activity against metastasis.Despite these findings, more studies with different cell lines are required to explore other pathways which could contribute to the control of the broad spectrum of TNBC.

Figure 1 .
Figure 1.Schematic representation of the metastatic process in triple negative breast cancer (TNBC)and its inhibition by non-platinum metal-based complexes (NPMBC).Metastasis is a sequential and interrelated multi-step process that consists of four main essential steps: migration to adjacent tissue after detachment from a primary tumor; local invasion of cancer cells into adjacent tissue; transendothelial migration of cancer cells into vessels (intravasation); transport of cancer cells through the circulatory system with extravasation of them in a secondary organ (brain, lung, liver, bone).The antimetastatic effect of NPMBC has been associated with their ability: to inhibit matrix metalloproteinases

Figure 1 .
Figure 1.Schematic representation of the metastatic process in triple negative breast cancer (TNBC)and its inhibition by non-platinum metal-based complexes (NPMBC).Metastasis is a sequential and interrelated multi-step process that consists of four main essential steps: migration to adjacent tissue after detachment from a primary tumor; local invasion of cancer cells into adjacent tissue; trans-endothelial migration of cancer cells into vessels (intravasation); transport of cancer cells through the circulatory system with extravasation of them in a secondary organ (brain, lung, liver, bone).The anti-metastatic effect of NPMBC has been associated with their ability: to inhibit matrix metalloproteinases (a); to inhibit mesenchymal markers such as vimentin (b); to interfere with the function of integrins (c); and to modify the immune/inflammatory response (d,e).

Table 2 .
Non-platinum metal based complexes (NPMBC) inhibit the in vitro and in vivo growth of triple-negative breast cancer (TNBC) cells by inhibiting tumor cell proliferation.

Table 3 .
Non-platinum metal based complexes (NPMBC) inhibit the in vitro and in vivo growth of triple-negative breast cancer (TNBC) cells by inducing apoptosis.

Table 4 .
Non-platinum metal based complexes (NPMBC) inhibit the in vitro and in vivo growth of triple-negative breast cancer (TNBC) cells by inducing autophagic cell death.

Table 5 .
Non-platinum metal based complexes (NPMBC) inhibit the in vitro and in vivo growth of triple-negative breast cancer (TNBC) cells by inhibiting migration, invasion and/or metastasis.