Background
Epithelial ovarian cancer is the fifth most common cause of cancer-related death in women and the most lethal gynecologic malignancy due to late stage diagnosis and upfront or eventual resistance to chemotherapy. Epithelial ovarian cancer is responsible for approximately 21,290 new cases and 14,180 deaths in 2015 in the United States [
1]. Despite new treatment modalities and improvements in diagnostic methods and surgical techniques, only a marginal improvement in survival has been achieved in the last 15 years and most patients will eventually have recurrence and succumb to their disease [
2]. Currently, optimal surgical debulking followed by platinum-based chemotherapy is the backbone of the ovarian cancer treatment; however, novel treatment modalities with less toxicity are urgently needed.
Nanotechnology is defined as the science involved in the design, synthesis, characterization, and manufacturing of materials at the nanoscale level [
3]. There has been a rapid growth in the nanotechnology field towards the development of nanomedicine products to improve therapeutic strategies against cancer [
4]. These nanomedical agents have been shown to improve the pharmacokinetic and pharmacodynamic properties of conventional treatment agents and may enhance the efficacy of existing anticancer compounds with less toxicity [
4]. Cerium is a rare earth metal and when combined with oxygen, can adopt a fluorite crystalline lattice structure. Nanoceria (NCe) particles can reversibly bind oxygen and shift oxidation states (Ce
3+/Ce
4+) depending on the environment [
5]. This interconversion of oxidation states gives rise to a large number of oxygen vacancies on the surface of NCe and is the key property that makes it a potent antioxidant agent. Due to its strong antioxidant properties, we have tested NCe as a therapeutic agent both in vitro and in vivo in ovarian cancer cells and showed that NCe inhibited the reactive oxygen species (ROS), vascular endothelial growth factor induced proliferation and capillary tube formation of endothelial cells. [
6]. NCe also attenuated cell migration and invasion without affecting the cell proliferation in ovarian cancer cell lines [
6]. Additionally, in vivo treatment of NCe was associated with significant reduction in tumor growth and accompanied by decreased proliferation and attenuation of angiogenesis in a nude mouse model of ovarian cancer. [
6]. NCe has also shown anticancer activity in other types of cancers including cutaneous squamous [
7], colon [
8] and pancreatic cancer [
9] models. Recently, it has been demonstrated that NCe in cancer cells associated with acidic conditions prefers to act as a ROS activator, resulting in accumulation of H
2O
2 [
9]. At acidic pH, NCe becomes an efficient superoxide dismutase mimetic compared to normal pH and converts superoxide radicals to H
2O
2. On the other hand, at acidic pH, NCe cannot scavenge H
2O
2 and eventually results in accumulation of H
2O
2 in cancer cells. This switch from acting as a superoxide dismutase or catalase mimetic to H
2O
2 accumulation has been attributed to the acidity dependent surface valence states of NCe (Ce
3+ or Ce
4+) [
7]. This increased generation of ROS may also be one of the mechanisms of NCe’s antitumor effect.
Folate is a basic component of cell metabolism, DNA synthesis and repair and essential for both normal and tumor cells [
10]. Two cellular folate uptake pathways have been identified: transmembrane reduced folate carrier and membrane-found folate receptors (FR) [
11]. Interestingly, normal cells utilize reduced folate carrier as a predominant way for folate uptake; however, FR is poorly expressed or absent in normal cells and strongly expressed in cancer cells [
11]. FRs capture folate from outside of the cell and the engulfed endosome is transported to the intracellular compartment [
10]. Three isoforms of FRs have been identified so far and these receptors have been the focus of cancer therapy. Increased expression of FR-α has been described in various tumor tissues, including ovarian, endometrial and breast cancer [
12], and higher FR-α expression was found to be associated with poor chemotherapy response and survival outcome in ovarian cancer [
13]. Monoclonal antibodies against FR-α, namely farletuzumab, combined with carboplatinum and taxanes was shown to increase the response rate in platinum-sensitive ovarian cancer patients [
14], but in another study in platinum-resistant/refractory patients, combination of farletuzumab with weekly paclitaxel did not reach its endpoint [
15]. Not only were monoclonal antibodies against FR- α investigated for treatment, but cellular entry process of folate was also utilized for targeted drug delivery in ovarian cancer by means of linking folate with chemotherapeutic agents. A combined treatment of vintafolide (EC145), a conjugate of folate with desacetylvinblastine monohydrazide, with liposomal doxorubicin was demonstrated to improve progression free survival over standard therapy in a randomized trial of patients with platinum-resistant ovarian cancer [
11].
In the current study, we investigated the preclinical efficacy of targeted delivery of NCe by its conjugation to folic acid (NCe-FA) alone and in combination with cisplatinum in inhibiting ovarian cancer growth in vitro and in vivo.
Discussion
In recent years, there has been a tremendous amount of data accumulation and understanding of tumor biology that could cause a paradigm shift in the treatment of cancer. Conventional chemotherapy still constitutes one of the main strategies for cancer treatment; however, it lacks the specificity against cancer cells and is associated with significant toxicity. Nanotechnology has recently been added to the treatment armamentarium with the aim to expand the horizons of cancer treatment. Nanoparticles have been shown to have a potential for early detection of cancer [
42], effective drug delivery that preferentially accumulates the drugs in tumor cells [
43] and potentiate the dose deposit selectively in irradiated tissues [
44]. In the current study, we showed that cerium nanoparticles conjugated with folic acid had a better cellular uptake as folic acid increased the concentration of NCe in ovarian cancer cells compared to NCe alone, resulting in better tumor inhibition and further enhancement of the tumoricidal effect of cisplatinum.
Cancer cells are highly dependent on folate for replication, and they even develop their own receptors that are not overexpressed on noncancerous cells, which makes it an excellent avenue for targeted anticancer treatment [
45,
46]. We previously showed that NCe has significant anticancer effect on ovarian cancer cell lines secondary to its antioxidant and antiangiogenic properties [
6]. We exploited this differential expression of folic acid receptors on cancer and somatic cells and demonstrated that conjugation of NCe with folic acid potentiated the NCe's therapeutic effect on ovarian cancer. One of the mechanisms for the higher efficacy might be higher intracellular accumulation of folic acid tagged particles. NCe-FA had significantly increased uptake by the cells, indicating that the conjugation with folic acid indeed enabled NCe to specifically target the cancer cells.
Tumor angiogenesis is essential for delivering oxygen and other nutrients to the growing tumor and is characterized by either excess production of pro-angiogenic signals or lack of angiogenesis inhibitors [
47‐
49]. Recently, inhibition of tumor angiogenesis has become a clinical anticancer strategy and only a few new drugs have been approved by the U.S. Food and Drug Administration for ovarian cancer since 2006, including an anti-angiogenic agent, bevacizumab [
50]. In our previous study, NCe was found to have significant antiangiogenic properties that is independent of VEGF production in vitro and in vivo
. We speculated that this might be secondary to inhibition of new vessel formation by targeting endothelial cells. In the current study, we examined the microvessel density in each group and found a significant reduction in formation of new vessels in NCe-FA and NCe-FA/cisplatinum treated groups compared to the untreated group as evident from decreased expression of CD31, a marker for endothelial cells. However, the NCe-FA and cisplatinum combination and cisplatinum alone groups showed similar CD31 density despite the fact that a lower tumor burden score was achieved with NCe-FA and cisplatinum treatment. Although the vessel density was similar, there might be a possibility that the integrity of the vessels may be more compromised in the combination group, resulting in nonfunctional vasculature, hence more reduced tumor growth. Additionally, NCe-FA also reduced the expression of the EMT marker, vimentin [
34], indicating its ability to maybe limit ovarian tumor metastasis. This supports our observation of reduced tumor nodules on the abdominal organs in the treated groups.
NCe particles have a dual effect on ROS generation that is mainly dependent on the pH of the environment [
7]. In the previous study, we showed that NCe (with higher Ce
3+; ~62 %) has significant antioxidant properties and decreased the ROS generation in A2780 cell lines in vitro. However, in the current study, a different formulation of NCe (with lower Ce
3+ [~24 %] as compared to the study published earlier) and NCe-FA conjugation increased the ROS. This might be secondary to the pH of the cancer cells. Wason et al. suggested that NCe acts primarily as a producer of H
2O
2 in (acidic) a cancer environment and a scavenger of H
2O
2 in (neutral) normal tissues [
9]. In particular, NCe converts superoxide radicals to H2O2; however, in an acidic condition pH suppress NCe catalase mimetic activity, which causes accumulation of H
2O
2 within the cells. In the presence of Fe ions, H
2O
2 can go through Fenton mediated reaction and might generate reactive radicals. Chemotherapy agents, such as anthracyclines and platinum complexes, also generate ROS and cause chemotherapy associated oxidative stress [
51]. This eventually decreases cell proliferation [
51]. The combination of NCe-FA and cisplatinum showed the strongest positive expression of 4 hydroxynonenal (oxidative stress marker) as they both increased the ROS production and eventually caused oxidative stress. These data are in accordance to the previous publication where NCe was shown to induce ROS generation in squamous carcinoma cells [
7] and pancreatic cancer cells [
9].
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
MH carried out the in vitro cytotoxicity and animal studies and participated in writing of manuscript; S Das, AG and SS synthesized and characterized the nanoparticle and its conjugation with folic acid; S Das, AG and SS wrote the nanoparticle synthesis and characterization parts of the manuscript, IM performed the immunohistochemistry and wrote the manuscript, ZA-W, CT, JC and SD carried out part of mouse studies and autopsies; AM participated in data analysis and manuscript writing; SS, SG and RR conceptualized the work, collected and interpreted the data. RR coordinated the study. All authors read and approved the manuscript.