*4.4. Oleuropein and Cancer*

Recently HY et al. [113] found that inhibition of autophagy in a Triple-Negative Breast Cancer cell line promoted migration and invasion, as demonstrated by exposition with Hepatocyte Growth Factor (HGT), or 3-methyladenine, an inhibitor of autophagy. On the contrary, the co-treatment with HT or Ole significantly suppressed HGF or 3-MA induced cell migration and invasion, by reversing LC3 II/I and Beclin-1 downregulation, and p62 upregulation.

Increased autophagy seems to be a defensive mechanism against treatment with doxorubicin (DXR). Papachristodoulou et al. [114] demonstrated in prostate cancer cells, that Ole is capable of lowering the cytotoxic dose of DXR significantly, without losing its anti-proliferative effect, via an induction of autophagy (see: Section 7. Chemiotherapy potentiation by Oleuropein).

These findings contribute to demonstrate the healthful actions of oleuropein against pathologies involving autophagy dysfunction, acting as an autophagy enhancer throught different mechanisms, and sugges<sup>t</sup> its potential use as a nutraceutical agen<sup>t</sup> in several NCDs.

## **5. Oleuropein as Anti–Amyloid Tool**

Many neurodegenerative pathologies, among which the most common are AlzD and PD, together with T2DM, are amyloid diseases (AD), and belong to the NCD group. In general, AD are diseases that are potentially fatal, defined by the occurrence of deposition of insoluble fibrillar polymeric material, grown from misfolded proteins (amyloid) in several organs. The core of these amyloids is made of unbranched polymeric fibrils of characteristic protein or peptides, typical for each disease, such as Aβ peptides for AlzD, α-synuclein for PD, amylin (hIAPP) for T2DM, and transthyretin (TTR) for familial amyloid cardiomyopathy [115–117]. Amyloidogenic proteins are characterized by β-sheet conformation, and share a common pathway of fibril formation. This latter is a complex process that involves the formation of an intermediate (soluble) oligomer form, following insoluble protofibril growth. Recently some authors have demonstrated that the cytotoxicity of different amyloidogenic proteins is due to soluble, intermediate oligomeric species, rather than to insoluble fibrillary amyloids [118]. Their cytotoxicity involves the disruption of calcium homeostasis, destabilization of membranes, ROS production, and apoptosis induction, all factors that determine cell suffering and death [119]. Interestingly, neurodegenerative diseases may extend outside the central nervous system (CNS), and can also involve the gastrointestinal tract (GI). Indeed, the same protein aggregates are present both in the enteric nervous system (ENS) and the CNS, leading to the hypothesis that the disease may start in the ENS and then spread retrogradely toward the CNS, or vice versa, and suggests that it may spread through a prion-like diffusion of misfolded protein accumulation, due to an imbalance between their production and clearance by autophagy systems [120].

So, the research of compounds interfering with aggregation of amyloid proteins is recognized as a valuable approach to build new therapeutic molecules. This is true expecially for AlzD, the most common form of dementia-related neurodegenerative disease among the elder people (aged 60 years and over), marked by a progressive decline in cognitive function and memory. Aggregates of Aβ peptides and neurofibrillary tangles of hyperphosphorylated tau proteins occurring in hypothalamic and cortical neurons are typical signs of this disease. PD is the second most common neurodegenerative disease, and is characterized by degeneration of dopaminergic neurons in the substantia nigra pars compacta due to deposition of intracellular inclusions known as LBs, the major component of which is α-synuclein. Although the clinical pathologies of these diseases have been described a long time ago, today there are drugs available only effective in reducing the symptoms of these diseases. The deposition of senile plaques and LBs in neuronal cells induces chronic stress, including oxidative stress and activation of microglial cells for the release of several pro-inflammatory cytokines, chemokines, and ROS, that are the major cause of these disorders [121].

OleA has been found to decrease toxic oligomers formation in vitro experiments of Aβ peptide and α-synuclein amyloid aggregation, as well as to promote fibril and plaque disaggregation [122–124]. These actions reflect its beneficial effects against amyloid toxicity to cultured cells [122] and in transgenic model organisms [125,126]. Using a simplified model of AlzD expressing human Aβ peptide, Diomede et al. [125] found that the larvae of a transgenic strain CL2006 of *Caenorhabditis elegans* fed with OleA, showed in the cytoplasm of muscle cells of the body wall, a reduction of Aβ plaque deposits, a lower content of toxic Aβ oligomers, a marked decrease of paralysis, and an increase of life expectancy compared with untreated animals.

hIAPP is a peptide hormone co-secreted with insulin by pancreatic β-cells, and fibrillar deposits of hIAPP amyloid aggregates in islets of Langerhans are a well known of T2DM. hIAPP aggregation together with oxidative stress (via NADPH-oxidase) leads to hIAPP toxicity, and plays a pivotal role in T2DM pathogenesis [127,128]. Rigacci et al. [74] showed that OleA occurrence, during the aggregation process, drove the formation of structurally different aggregates by hindering their binding with the membrane cells, resulting in a decrease of membrane damage, and protection of rat insulinoma cells against aggregate cytotoxicity, the main aspect responsible for cell sufferance. Similar results were obtained by Leri et al. [129] that extend the above finding to the OleA/TTR system, the latter is involved in a subset of familial or sporadic amyloid diseases including senile systemic amyloidosis (SSA), familial amyloid polyneuropathy and cardiomyopathy (FAP/FAC), for which no effective therapy has ye<sup>t</sup> been found. This polyphenol was able to interfere with TTR fibril assembly and to promote mature fibril disruption. In this study, OleA protection against amyloid TTR toxicity, tested on in vitro HL-1 cells, resulted from stabilizing an oligomer-like intermediate that interacts with the plasma membrane without altering its integrity. In addition, OleA was also found to be able to disassemble pre-formed TTR mature fibrils into the same non-toxic oligomer-like intermediates [129].

Another interesting aspect of the anti-cytotoxic action of OleA, in the contest of amyloydosis, was reported by Leri et al. [130]. Using another amyloidogenic protein, a variant of human β2-microglobulin (β2m) [131–133] a 99 residue-long human protein belonging to the major histocompatibility complex class I (MHC I) associated with a familial form of systemic amyloidosis, they showed that it exhibited an enhanced amyloidogenic tendency to aggregate in vitro with respect to the wild-type protein. In this study, they found that OleA modified not only the conformational and biophysical properties of the amyloid fibrils, favoring the appearance of non-toxic aggregates, but also modified the cell bilayer surface properties, decreasing aggregate interaction with the plasma membrane of exposed cells, and enhancing cell resistance against the toxic effects of the aggregates.

All these data sugges<sup>t</sup> a possible use of OleA as a novel and promising pharmacological tool, acting directly on amyloid formation *via* the protein self-assembly pathway, for prevention and therapy of systemic amyloidosis.

#### **6. Oleuropein As an Anticancer Agent**

Current protocols for cancer treatment are dependent on the condition of the tumor at time of diagnosis. If diagnosed early, the tumor mass may be removed by surgery, but if it has spread to lymph nodes, surgery will be more intensive, and chemotherapy and immunotherapy will likely be added to the treatment. Up-to-now, chemotherapy and immunotherapy represent a promising route for a more e ffective, life-saving cure for most human cancers. Despite advancements in these therapies, many patients with metastatic lesions still face a significant mortality risk. Furthermore, chemotherapy and immunotherapy may result in patient resistance, and generate host side e ffects. Therefore, new strategies that target cancer cells and also reduce resistance and patient side e ffects, may help the development of new treatments. Thus, the combination of conventional treatment with biological agents (so-called complementary therapy) may enhance the e fficacy of treatment, and reduce drug resistance. In addition, complementary therapy may provide a reduction in side e ffects and improve the overall quality of life of patients during therapy.

Oleuropein may contribute to therapy in several ways, including its inhibitory role in some crucial cancer cell activities, and these are summarized in the Figure 3.

**Figure 3.** Effect of oleuropein (Ole) on the factors contributing to cancer development.

#### *6.1. Pro-Apoptotic and Anti-Proliferative E*ff*ects of Oleuropein*

Bouallagui et al. [134], investigating Ole-enriched extract and its derivative HT, found a consistent inhibition of the proliferation of luminal breast cancer cells (MCF-7 cells), which were arrested in G0/G1 phase. The probable mechanism of growth-arrest was Cyclin D1 inhibition by these polyphenols. Han et al. [135] also reported in the same cancer cells, a blocking of the transition from G1 to S phase, caused by HT and OleA. Elamin et al. [136] was able to extend the use of Ole to the less responsive

basal-like breast cancer cells (MDA-MB-231 cells), and demonstrated its ability to abrogate NF-kB expression, a well-known transcription factor involved in the control of many genes driving cancer development and progression, e.g., inflammation, immune reaction, proliferation, and apoptosis. This last finding was also confirmed by Liu et al. [137].

Further exposing BPH-1 normal prostate cells, androgen-sensitive LNCaP, and androgeninsensitive DU145 prostate cancer cells to Ole, promoted an anti-oxidant action in normal cells, whereas in cancer cells, it induced pro-oxidant and anti-proliferative effects, suggesting a quite cancer-specific effect [138].

It is also known that an Ole-enriched diet prevents azoxymethane-induced pre-neoplastic lesions of the colon, reducing dysplasia and DNA damage [139].

Cardeno et al. [140] showed that Ole and HT significantly inhibits HIF-1a and promotes the expression of p53 in HT-29 human colon adenocarcinoma cells, as a critical change to limit proliferation, and induce apoptosis.

Additional evidence suggests that one of the best inhibitors of human colon carcinoma cells is the HT oleate form, suggesting that long chain fatty acids, such oleate, may facilitate Ole activity [141].

Ole from Corregiola leaf extracts expresses a very high level of anti-proliferative activity on pancreatic cancer cells (MiaPaCa-2 cells), opening up a possibile treatment for one of the most aggressive human cancers.

Samara et al. [142] studied 51 analogs of Ole on several human cancer cells, and found that analog 24, non-toxic for normal cells, expresses the highest level of inhibitory activity in vitro (human colon cancer cells HCT-116, human cervical carcinoma cells HeLa, MCF-7 cells) as well as in vivo (B16-F10 mouse melanoma cells). Of a particular importance was the finding that Ole analog 24 expresses a promotion of natural immune responses, from Natural Killer cells and Limphokine-activated Killer cells.

Several authors showed Ole promoted apoptosis in cancer cells, like HeLa cells [143], HepG2 human hepatoma cells [144], SH-SY5Y human neuroblastoma cells [145], and HCT116 cells [146]. Taken together, the above researches provide significant insights into the contribution of Ole in treating cancer cells, disclosing several targets of its pro-apoptotic activity, such as activation of the JNK pathway, suppression of PI3K/AKT signaling, and activation of caspase-9 and 3 gene expression.

Additionally, pro-apoptotic activity of Ole has been seen against HL60 human promyelocytic leukemia cells [147].

#### *6.2. Multiple Checkprotein of Transcription of Oleuropein*

Further indications of the multiple targets of Ole activity on cancer cell machinery has emerged from the following contributions. Sirianni et al. [148] demonstrated that HT and Ole inhibit estrogen-dependent proliferation in MCF-7 cells, affecting extracellular-regulated kinases 1/2 (ERK 1/2) of the mitogen activating protein kinase family.

More-recently, Momtaz et al. [149] has shown the influence of Ole on melanoma. Melanoma is certainly one of the most aggressive skin cancers, due to its progression to drug resistance and its organ dissemination. Among the signalling pathways able to control melanoma development, janus kinase signal transducers and activators of transcription (JAKs/STATs) are the most critical, and polyphenols are able to target the most active, STAT3.

Furthermore, Ole was found to affect T-type Ca2+ channels altering the dynamics of intracellular Ca2+ in mesothelioma cancer cells. T-type calcium channels are low voltage channels, which open up when the membrane is depolarized, and calcium enters into various cells with different responses. On the other hand, mesothelioma is a cancer with a very poor prognosis, with an urgen<sup>t</sup> need for new targeted therapies [150].

In MCF-7 cells, Ole exhibits an additional ability that extends its anti-cancer effects to the modulation of tumor suppressor genes, such as onco-miRNAs (miRNA-21 and miR-155) [151]. Researches by Tezcan G. et al. [152] serve to underline that Ole may modulate expression of miRNAs, miR-137, -145, and -153, in glioblastoma multiforme (GBM) cancer stem cells.

Bayat et al. [153], recently proved that Ole modulates epigenetic inhibiting histone deacetylases (HDAC2 and HDAC3). The use of HDAC inhibitors in cancer treatment represents a new approach to therapy, like reactivation of tumor suppressor genes. Although, this treatment causes side effects in the treated patients, Ole might have a role in some complementary therapies using HDAC inhibitors.

#### *6.3. Other Biological Activities of Oleuropein*

Ole was reported to express a potent inhibitory activity on tumor xenografts, disrupting actin filaments, thus abrogating proliferation, motility, and invasiveness [154]. The several cancer cell lines used in the experiments is an additional parameter of importance in this latter study (GMB cells, renal adenocarcinoma cells, breast cancer cells, melanoma cells, and colorectal adenocarcinoma cells).

Starting from the epidemiological observation that obesity is associated with an increased risk of developing many cancers, and metastatic dissemination represents the leading cause of mortality of cancer patients, a study by Song et al. [155] suggests that Ole inhibits tumor growth and lymph node metastasis in mouse melanoma cells, abrogating angiogenesis and lymphangiogenesis through the reduction of the peroxisome proliferator-activated receptor γ and the infiltration of M2 macrophages, both responsible for the secretion of angiogenesis and lymphangiogenesis inducers, VEGFA and D, respectively.

Evidence for the role of Ole in promoting the differentiation of K562 multipotent leukemia cells toward a monocyte lineage, was reported by Samet et al. [156]. Further anti-cancer/pro-apoptotic activity of Ole on leukemic cells, like HL60 human promyelocytic leukemia cells, was reported by Anter et al. [147].

The anticancer effect of Ole on glioma cells was characterized by a decrease of matrix MMP-2 and -9 activity, leading to a significant reduction in their ability to invade extracellular tissues [157]. In accord with this last observation, Ole was found to promote a reduction in the metastatic ability of breast cancer cells that was ascribed to a MMP-2 and -9 decrease, strengthened by a promotion of tissue inhibitors of MMP (TIMP) 1,3,4 [158]. It is known that a balance of MMP and TIMP regulates cancer cell invasion and dissemination.

## **7. Chemiotherapy Potentiation by Oleuropein**

Due to the several anticancer properties of oleuropein, including anti-proliferative and pro-apoptosis activity, it was investigated whether it might represent an effective agen<sup>t</sup> for complementary cancer therapy. In addition, it is well known that most anticancer drugs induce side-effects and toxicity in tumor-bearing patients, which oleuropein might be able to overwhelm.

A study reported by Papachristodoulou et al. [114] shows that co-treatment with doxorubicin and Ole affected cell proliferation of PC3 prostate cancer cells in an additive manner, even using a very low dose of doxorubicin. These authors also showed that the co-treatment did not modify cell cycle distribution and apoptosis of these cells in a significant way, but was able to induce a significant promotion of autophagy. Tumor cells are characterized by high energy use, even during starvation, when autophagy may sustain mitochondrial functions. Thus, the autophagy-dependence of cancer cells discloses a new therapeutic target for Ole, as reported above. Moreover, Ole has been shown to protect against the cardiotoxicity of doxorubicin [159]; this is an additional ability that further promotes the use of oleuropein in cancer co-treatments.

GBM is considered one of the most malignant human cancers, and therapy is up-to-now only palliative. GBM expresses a capillary network which contributes to tumor expansion and invasion, for which a targeted therapy using a monoclonal anti-VEGA antibody, bevacizumab, might be effective. However, this treatment often promotes an enhanced aggressive phenotype in resistant GBM cells. Tezcan et al. [160] were able to demonstrate that Ole synergistically increases bevacizumab's anti-angiogenesis and anti-migration effects. Ole has the ability to promote the effects of bevacizumab, preventing VEGFA, MMP-2 and -9 activities. A large body of clinical and experimental evidence indicates a key role of PI3K/Akt/mTOR hyper-activation in GBM biology, which in turn, sustains cell

metabolism by promoting protein synthesis and suppressing autophagy, the major protein degradation pathway [161]. In our previous study, we showed that in BRAF melanoma cells, Ole a ffected cell proliferation by downregulation of the pAKT/pS6 pathway [162]. Further studies and validation are required, but it is likely that Ole, through its mTOR inhibition and autophagy induction, may help in the development of new therapeutic strategies against GBM.

A further histotype of cancer with no e ffective treatment is hepatocellular carcinoma (HCC), which forms metastases and develops drug resistance very early. Thus, an e ffective and low-toxicity therapy is required. Sherif et al. [163] has demonstrated that Ole greatly potentiates the reduction of MMP-7 gene expression in HepG2 cells induced by cisplatin. This ability was found to be instrumental to reduce the cancer promoting ability of nerve growth factor (NGF) on HepG2 cells. Indeed, mature NGF is secreted from its precursor form, pro-NGF, through MMP-7 proteolytic cleavage, and exerts a pro-survival e ffect on HCC cells. The co-stimulation of Ole with cisplatin also enhances caspase-3 gene expression in these cells, potentiating their apoptotic rate.

Thereafter, our laboratory [162] demonstrated that Ole enhances chemotherapy of BRAF melanoma cells, by downregulating the pAKT/pS6 pathway. Of a particular significance, the finding that Ole was able to promote the death e ffect of Everolimus, a mTOR inhibitor, in Vemurafenib-resistant BRAF melanoma cells, points to a possibility for its use in treating resistant melanoma cells. OleA also contributed to the cytotoxic e ffect of dacarbazine against BRAF melanoma cells. As in resistant melanoma cells, exposure to OleA was found to reverse trastuzumab resistance in HER2-overexpressing breast cancer cells [164].

In addition to chemotherapy, Ole has been proved to enhance the radiation sensitivity of nasopharyngeal carcinoma cells repressing mRNA-519d [165].

On the whole, oleuropein has attracted the attention of several researchers, due to its ability to discriminate between cancer and normal cells, inhibit proliferation, and promote apoptosis in several tumors, including those tumors which are considered the most aggressive, such as mesothelioma, GBM and melanoma. Finally, a real potentiating e ffect of oleuropein on standard chemotherapy has been demonstrated.
