metabolic and molecular studies in patients, animals and cell models (Polysen study)

Summary: Breast cancer is the most diagnosed among women and accounts for 15% of cancer deaths among the female world population. It has a multifactorial etiology, but only 10-15% are hereditary cases. This implies that modifiable risk factors are those that are mainly associated with its incidence. While obesity or alcohol abuse increase the risk of breast cancer, a higher consumption of plant foods could have a possible preventive effect. Numerous preclinical studies with phenolic compounds show a multitude of protective mechanisms of action against breast cancer. However, the evidence in humans is still scarce and controversial. This Doctoral Thesis includes an exhaustive and critical review of the reasons behind this still inconsistent evidence, including the lack of i) studies in patients with breast cancer that relate the beneficial effects with phenolic compounds, ii) preclinical studies with adequate methodologies to extrapolate its results to a clinical context, and iii) studies that evaluate the bioavailability and distribution in systemic human tissues. Considering the above, this Thesis has had as its main objective to identify which compounds (molecular forms and concentrations) reach the tumorous (TTM) and healthy (SST) breast tissue in patients with breast cancer after the consumption of a mixture of plant extracts, rich in phenolic, representative of the diet. This objective would serve to address mechanistic in vitro studies, more representative of the in vivo situation, on the chemopreventive effect of physiologically relevant metabolites in cellular models of breast cancer. To address these objectives, a clinical study was carried out in patients with breast cancer, where the metabolic profile of different phenolic compounds and their metabolites in TTM and SST, plasma and urine of these patients was characterized, after consuming a mixture of plant extracts. In addition, the pharmacokinetics in plasma and mammary tissue of phenolics and derived metabolites were studied in an animal model (Sprague Dawley rats) after the consumption of this mixture. The clinical study allowed the identification of 27 phenolic metabolites and 6 methylxanthines in TTM of patients with breast cancer, most of the molecular forms detected in TTM, being Phase-II conjugates (glucuronides and sulfates). The animal study allowed us to know kinetic parameters in plasma and breast tissue of phenolics and their metabolic derivatives, and served to identify that fasting prior to surgery in patients can qualitatively and quantitatively underestimate the distribution of phenolic metabolites in breast tissue. The metabolites that reach the TTM did not exert antiproliferative effect, nor estrogenic / antiestrogenic activity, at short exposure times in different cellular models of breast cancer. Subsequent studies revealed that Phase-II metabolism is critical to hinder / abolish the biological activity of metabolites. However, resveratrol and its Phase-II metabolites, detected in human TTM, did exert a long-term chemopreventive effect by inducing cellular senescence (dependent on the p53 / p21 pathway) in the MCF-7 tumor line (p53- wild type) but not in MDA-MB-231 (p53-mutated). The latter encourages investigating the chemopreventive effect of other phenolics detected in TTM by inducing senescence, which would be plausible with chronic consumption of foods rich in phenolics. In general, this Thesis has contributed to understanding which phenolic compounds and metabolites reach TTM in patients with breast cancer after the consumption of plant foods, and what possible role these compounds could play against cancer. Furthermore, this Thesis reinforces the concept of “first in vivo, then in vitro”, and establishes a possible sequence of studies to follow to characterize the anticancer activity of dietary components in systemic tissues.
Breast cancer is the most commonly diagnosed cancer in women worldwide. Overall, this disease accounts for 15% of cancer deaths among the female population. Breast cancer has multifactorial etiology, but only 10-15% of cases are hereditary. This means that the vast majority are caused by modifiable factors. Thus, it has been described that while obesity or alcohol consumption increase the risk of breast cancer, the higher consumption of phenolic-rich plant foods could be related to a possible preventive effect. Although many preclinical studies have reported numerous mechanisms of action of phenolics that would explain their protective role against breast cancer, however, evidence remains still scarce and controversial in humans. This Doctoral Thesis reviews comprehensively and critically the reasons that are behind this inconsistent evidence, including i) the low number of clinical trials that report beneficial effects of phenolics against breast cancer, ii) the big gap between preclinical and clinical research due to the assay of non-physiological conditions, and iii) the lack of studies that evaluate the bioavailability and distribution of phenolics and derived metabolites in systemic human tissues.
Considering the above, this Thesis has aimed to identify which compounds (molecular forms and concentrations) can reach malignant (MMT) and normal (NMT) mammary tissues of breast cancer patients after consuming a mixture of phenolic-rich plant extracts, representative of a dietary context. This knowledge could help to design mechanistic studies, illustrative of the in vivo situation, to evaluate the potential chemopreventive effect of physiologically relevant phenolic-derived metabolites using different breast cancer cell models.
To address this objective, we conducted a clinical trial with breast cancer patients to characterize the metabolic profile of different dietary phenolics and their derived metabolites in MMT and NMT, plasma, and urine of patients after consuming a mixture of plant extracts. Furthermore, the plasma and tissue pharmacokinetics of phenolics and derived metabolites after consuming the same mixture of plant extracts were evaluated in an animal model (Sprague Dawley rats).
The clinical trial allowed identifying 27 phenolic-derived metabolites and 6 methylxanthines in MMT from breast cancer patients. Phenolic-derived Phase-II conjugated metabolites (mainly glucuronides and sulfates) were the main molecular forms detected in MMT and NMT. Furthermore, the animal study revealed the kinetics of phenolics and derived metabolites in plasma and mammary tissues, and determined that fasting before surgery qualitatively and quantitatively was a critical issue to underestimate the distribution of phenolic-derived metabolites in systemic tissues, including mammary tissue.
The in vitro assays showed that phenolic-derived metabolites occurring in MMT, even at much higher concentrations, did not exert antiproliferative, neither estrogenic/antiestrogenic, effects in different breast cancer cell models after short-term exposure. Further cell studies demonstrated that Phase-II conjugation was critical to hamper the chemopreventive effects of phenolics and derived metabolites in breast cancer cell models.
However, resveratrol and their Phase-II metabolites, as detected in human MMT, induced cellular senescence (dependent on the p53/p21 pathway) in MCF-7 breast cancer cells (wild-type p53), but not in MDA-MB-231 cells (mutant p53), after long-term exposure. These results open new research scenarios to explore possible chemopreventive effects of other relevant phenolic-derived metabolites detected in MMT. These beneficial effects against breast cancer through cellular senescence induction would be plausible with chronic consumption of phenolic-containing foods.
Overall, this Thesis has mainly contributed to understanding what phenolics and derived metabolites reach the MMT in breast cancer patients after consuming plant foods, and what possible protective role against this cancer they could play. Besides, this Thesis claims for the concept ‘first in vivo, and then, in vitro’, and establishes a possible sequence of studies to be followed for characterizing the cancer chemopreventive activity of dietary compounds in systemic tissues.

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