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Oleocanthal and oleocanthal-rich olive oils induce lysosomal membrane permeabilization in cancer cells

Oleocanthal is a phenolic compound found in varying concentrations in extra virgin olive oil Oleocanthal has been shown to be active physiologically, benefiting several diseased states by conferring anti-inflammatory and neuroprotective benefits. Recently, we and other groups have demonstrated its specific and selective toxicity toward cancer cells; however, the mechanism leading to cancer cell death is still disputed. The current study demonstrates that oleocanthal, as well as naturally oleocanthal-rich extra virgin olive oils, induced damage to cancer cells’ lysosomes leading to cellular toxicity in vitro and in vivo. Lysosomal membrane permeabilization following oleocanthal treatment in various cell lines was assayed via three complementary methods. Additionally, we found oleocanthal treatment reduced tumor burden and extended lifespan of mice engineered to develop pancreatic neuroendocrine tumors. Finally, following-up on numerous correlative studies demonstrating consumption of olive oil reduces cancer incidence and morbidity, we observed that extra virgin olive oils naturally rich in oleocanthal sharply reduced cancer cell viability and induced lysosomal membrane permeabilization while oleocanthal-poor oils did not. Our results are especially encouraging since tumor cells often have larger and more numerous lysosomes, making them especially vulnerable to lysosomotropic agents such as oleocanthal.


Introduction
Olive oil has been consumed by humans for millennia and is frequently 49 associated with health-related properties. The Mediterranean diet and olive oil 50 consumption in particular are correlated with lower cancer incidence and mortality (1-3).

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A meta analyses of nineteen observational studies performed between 1990 and 2011 52 that included approximately 35,000 individuals found that olive oil intake is inversely 53 related to cancer prevalence (4). More recently, a randomized trial found that women 54 who adhered to a Mediterranean diet supplemented with extra virgin olive oil (EVOO) 55 had 62% less invasive breast cancer incidence than a control group that was advised to 56 restrict dietary fats (5). These studies, however, did not distinguish between the 57 protective effects of EVOO's triglycerides and its phenolic components. Furthermore, to 58 the best of our knowledge, no controlled study tested the effect of high phenolic EVOO 59 on cancer. (6). A few years later, it was reported to be the primary agent that conveys strong 63 stinging sensation at the back of the throat when ingesting certain EVOOs (7). In 2005, 64 Beauchamp and colleagues published the first paper to refer to this compound as   forms, for purposes of invasion, angiogenesis, and progression (24, 25). The increased 108 reliance on lysosomal processes might also represent an Achilles' heel for cancer. As 6 114 cells and triggers apoptotic death mechanisms, whereas high levels of LMP kills cells 115 rapidly and directly as a form of necrosis.

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In this report, we demonstrate oleocanthal's ability to induce severe LMP in a 117 variety of cancer cells lines, leading to rapid necrotic cell death in vitro and shrinkage of 118 tumors and extension of lifespan in an in vivo mouse model for pancreatic 119 neuroendocrine tumors (PanNET). Strikingly, we were also able to replicate the 120 beneficial effects of purified oleocanthal by treating cells with EVOOs that naturally 121 contain high levels of oleocanthal.         (Fig 1B) -indicating that the cell death induced by oleocanthal is rapid. 266 We previously reported that oleocanthal-induced cell death is due to a necrotic 267 mechanism, whereas other groups have reported the mechanism of death to be 268 apoptotic (15, 16).

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To further establish the mechanism of cell death, we performed a well-       stability as compared to vehicle (Fig 2D). This indicates that oleocanthal does not act 342 directly as a membrane disrupting agent on lysosomes, but rather induces lysosomal 343 permeability only in a cellular context -likely through oleocanthal metabolites.

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The heat shock protein HSP70 is known to stabilize lysosomal membranes (28) 345 and in various models of LMP, HSP70 provides protection from subsequent cell death 346 (38). We, therefore, overexpressed HSP70 in PC3 prostate cancer cells and examined 347 the effect on oleocanthal-induced loss of cell viability. Indeed, oleocanthal-induced loss 348 of cell viability was partially rescued by HSP70 overexpression (Fig 2E), further 349 supporting a role for LMP as the cause of oleocanthal-induced cell viability.    Fig 3B).

399
To determine whether the tumors were smaller due to LMP induced cell death,       no oleocanthal had no effect on cell viability. Furthermore, by looking at cytosolic 513 cathepsin release, the EVOOs mechanism of promoting cancer cell death also involved 514 LMP, similar to the effects of purified oleocanthal.

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Many studies have linked consumption of EVOO with reduced incidence of 516 cancer (4), most significantly a randomized trial in which elevated EVOO in the diet led 517 to a 62% reduction in the incidence of breast cancer in Spain over a 5 year period (5).

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Data provided here link the cytotoxic effects of EVOOs to their level of oleocanthal. The 519 cytotoxic effects were due to the ability of oleocanthal to induce LMP and necrotic cell 520 death preferentially in cancer cells. Whereas pure oleocanthal can also have negative 521 effects on non-cancerous cells, EVOO is considered safe and healthy and, therefore, 522 could be both preventative as well as a potential treatment -as indicated by the 523 Spanish study (5). Since the apparent target for oleocanthal-induced necrosis is the 524 lysosome, the reason for the elevated sensitivity of cancer cells to oleocanthal could be 24 525 due to the increased size and fragility of the lysosomal compartment of cancer cells 526 (27). If the enlarged fragile lysosomal compartment (23, 41) is the reason for increased 527 sensitivity to oleocanthal, it is likely that EVOOs with high oleocanthal could be 528 preventative for many cancers -in addition to reduced breast cancer in Spain (5).

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Whether purified oleocanthal could be used therapeutically remains to be evaluated.