Ex Vivo Anti-Leukemic Effect of Exosome-like Grapefruit-Derived Nanovesicles from Organic Farming-The Potential Role of Ascorbic Acid.

Citrus fruits are a natural source of ascorbic acid, and exosome-like nanovesicles obtained from these fruits contain measurable levels of ascorbic acid. We tested the ability of grapefruit-derived extracellular vesicles (EVs) to inhibit the growth of human leukemic cells and leukemic patient-derived bone marrow blasts. Transmission electron microscopy and nanoparticle tracking analysis (NTA) showed that the obtained EVs were homogeneous exosomes, defined as exosome-like plant-derived nanovesicles (ELPDNVs). The analysis of their content has shown measurable amounts of several molecules with potent antioxidant activity. ELPDNVs showed a time-dependent antiproliferative effect in both U937 and K562 leukemic cell lines, comparable with the effect of high-dosage ascorbic acid (2 mM). This result was confirmed by a clear decrease in the number of AML blasts induced by ELPDNVs, which did not affect the number of normal cells. ELPDNVs increased the ROS levels in both AML blast cells and U937 without affecting ROS storage in normal cells, and this effect was comparable to ascorbic acid (2 mM). With our study, we propose ELPDNVs from grapefruits as a combination/supporting therapy for human leukemias with the aim to improve the effectiveness of the current therapies.

Nanovesicles from Organic Agriculture-Derived Fruits and Vegetables: Characterization and Functional Antioxidant Content.

Dietary consumption of fruits and vegetables is related to a risk reduction in a series of leading human diseases, probably due to the plants' antioxidant content. Plant-derived nanovesicles (PDNVs) have been recently receiving great attention regarding their natural ability to deliver several active biomolecules and antioxidants. To investigate the presence of active antioxidants in fruits, we preliminarily analyzed the differences between nanovesicles from either organic or conventional agriculture-derived fruits, at equal volumes, showing a higher yield of nanovesicles with a smaller size from organic agriculture-derived fruits as compared to conventional ones. PDNVs from organic agriculture also showed a higher antioxidant level compared to nanovesicles from conventional agriculture. Using the PDNVs from fruit mixes, we found comparable levels of Total Antioxidant Capacity, Ascorbic Acid, Catalase, Glutathione and Superoxide Dismutase 1. Finally, we exposed the nanovesicle mixes to either chemical or physical lytic treatments, with no evidence of effects on the number, size and antioxidant capacity of the treated nanovesicles, thus showing a marked resistance of PDNVs to external stimuli and a high capability to preserve their content. Our study provides for the first time a series of data supporting the use of plant-derived nanovesicles in human beings' daily supplementation, for both prevention and treatment of human diseases.

In vivo antiaging effects of alkaline water supplementation.

Telomeres length and telomerase activity are currently considered aging molecular stigmata. Water is a major requirement for our body and water should be alkaline. Recent reports have shown that aging is related to a reduced water intake. We wanted to investigate the effect of the daily intake of alkaline water on the molecular hallmark of aging and the anti-oxidant response. We watered a mouse model of aging with or without alkaline supplementation. After 10 months, we obtained the blood, the bone marrow and the ovaries from both groups. In the blood, we measured the levels of ROS, SOD-1, GSH, and the telomerase activity and analysed the bone marrow and the ovaries for the telomeres length. We found reduced ROS levels and increased SOD-1, GSH, telomerase activity and telomeres length in alkaline supplemented mice. We show here that watering by using alkaline water supplementation highly improves aging at the molecular level.

Moving the systemic evolutionary approach to cancer forward: Therapeutic implications.

We have previously presented a new Systemic Evolutionary Theory of Cancer (SETOC) based on the failure of proper endosymbiosis in eukaryotic cells. Here, we propose that the progressive uncoupling of two endosymbiotic subsystems (information and energy) inside the cell, as a consequence of long-term injuries, gives rise to alterations (i) in tissue interactions and (ii) in cell organization. In the first case, we argue that the impairment of both the coherent state and the synergy between intercellular communications underpins the onset of tissue dysplasia, that usually evolves towards cancer development. In the second case, we suggest that the rupture of endosymbiosis drives a sort of cell regression towards a protist-like entity represented by the concept of "de-emergence" postulated in our systemic evolutionary approach to carcinogenesis. This conceptual association of the cancer cell with a protist-like organism could support the development of novel cancer therapeutic approaches. To this end, we propose a paradigm shift in cancer pharmacology since: i) our knowledge of cancer pathophysiology as a complex system is insufficient, despite a vast knowledge of molecular mechanisms underlying cancer; ii) current cancer pharmacology deals only with microvariables (e.g. gene or protein targets), which do not account for the integrated pathophysiology of cancer, rather than with macrovariables (e.g. pH, membrane potential, electromagnetic fields, cell communications and so on) and mesovariables (between micro and macro), such as the interaction between various cellular components including cellular organelles. This paradigm shift should allow cancer pharmacology to move forward from molecular treatments (focusing on single targets) to modular treatments that consider cancer-related processes (i.e. inflammation, coagulation, etc.) or even to a sort of ecosystemic treatment addressing the whole functioning of the "cancer ecosystem". Examples of ecosystems treatment may be natural plant derivatives that act synergistically or pulsed electromagnetic fields which can act on particular biological processes in cancer cells. In addition, we need different working theoretical models on which to base new anticancer pharmacological approaches. Finally, we examine what value our systemic evolutionary approach could add to cancer treatments, in particular in liver cancer as a paradigm for developing potential applications.

Antitumor effect of combination of the inhibitors of two new oncotargets: proton pumps and reverse transcriptase.

Tumor therapy needs new approaches in order to improve efficacy and reduce toxicity of the current treatments. The acidic microenvironment and the expression of high levels of endogenous non-telomerase reverse transcriptase (RT) are common features of malignant tumor cells. The anti-acidic proton pump inhibitor Lansoprazole (LAN) and the non-nucleoside RT inhibitor Efavirenz (EFV) have shown independent antitumor efficacy. LAN has shown to counteract drug tumor resistance. We tested the hypothesis that combination of LAN and EFV may improve the overall antitumor effects. We thus pretreated human metastatic melanoma cells with LAN and then with EFV, both in 2D and 3D spheroid models. We evaluated the treatment effect by proliferation and cell death/apoptosis assays in classical and in pulse administration experiments. The action of EFV was negatively affected by the tumor microenvironmental acidity, and LAN pretreatment overcame the problem. LAN potentiated the cytotoxicity of EFV to melanoma cells and, when administered during the drug interruption period, prevented the replacement of tumor cell growth.This study supports the implementation of the current therapies with combination of Proton Pumps and Reverse Transcriptase inhibitors.

Effect of Modified Alkaline Supplementation on Syngenic Melanoma Growth in CB57/BL Mice.

Tumor extracellular acidity is a hallmark of malignant cancers. Thus, in this study we evaluated the effects of the oral administration of a commercially available water alkalizer (Basenpulver®) (BP) on tumor growth in a syngenic melanoma mouse model. The alkalizer was administered daily by oral gavage starting one week after tumor implantation in CB57/BL mice. Tumors were calipered and their acidity measured by in vivo MRI guided 31P MRS. Furthermore, urine pH was monitored for potential metabolic alkalosis. BP administration significantly reduced melanoma growth in mice; the optimal dose in terms of tolerability and efficacy was 8 g/l (p< 0.05). The in vivo results were supported by in vitro experiments, wherein BP-treated human and murine melanoma cell cultures exhibited a dose-dependent inhibition of tumor cell growth. This investigation provides the first proof of concept that systemic buffering can improve tumor control by itself and that this approach may represent a new strategy in prevention and/or treatment of cancers.

Intermittent high dose proton pump inhibitor enhances the antitumor effects of chemotherapy in metastatic breast cancer.

BACKGROUND: Acidity is a hallmark of malignant tumor, representing a very efficient mechanism of chemoresistance. Proton pump inhibitors (PPI) at high dosage have been shown to sensitize chemoresistant human tumor cells and tumors to cytotoxic molecules. The aim of this pilot study was to investigate the efficacy of PPI in improving the clinical outcome of docetaxel + cisplatin regimen in patients with metastatic breast cancer (MBC). METHODS: Patients enrolled were randomly assigned to three arms: Arm A, docetaxel 75 mg/m(2) followed by cisplatin 75 mg/m(2) on d4, repeated every 21 days with a maximum of 6 cycles; Arm B, the same chemotherapy preceded by three days esomeprazole (ESOM) 80 mg p.o. bid, beginning on d1 repeated weekly. Weekly intermittent administration of ESOM (3 days on 4 days off) was maintained up to maximum 66 weeks; Arm C, the same as Arm B with the only difference being dose of ESOM at 100 mg p.o. bid. The primary endpoint was response rate. RESULTS: Ninety-four patients were randomly assigned and underwent at least one injection of chemotherapy. Response rates for arm A, B and C were 46.9, 71.0, and 64.5 %, respectively. Median TTP for arm A (n = 32), B (n = 31), C (n = 31) were 8.7, 9.4, and 9.7 months, respectively. A significant difference was observed between patients who had taken PPI and who not with ORR (67.7 % vs. 46.9 %, p = 0.049) and median TTP (9.7 months vs. 8.7 months, p = 0.045) [corrected]. Exploratory analysis showed that among 15 patients with triple negative breast cancer (TNBC), this difference was bigger with median TTP of 10.7 and 5.8 months, respectively (p = 0.011). PPI combination showed a marked effect on OS as well, while with a borderline significance (29.9 vs. 19.2 months, p = 0.090). No additional toxicity was observed with PPI. CONCLUSIONS: The results of this pilot clinical trial showed that intermittent high dose PPI enhance the antitumor effects of chemotherapy in MBC patients without evidence of additional toxicity, which requires urgent validation in a multicenter, randomized, phase III trial. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT01069081 .

Proton pump inhibitor chemosensitization in human osteosarcoma: from the bench to the patients' bed.

BACKGROUND: Major goals in translational oncology are to reduce systemic toxicity of current anticancer strategies and improve effectiveness. An extremely efficient cancer cell mechanism to avoid and/or reduce the effects of highly cytotoxic drugs is the establishment of an acidic microenvironment, an hallmark of all malignant tumors. The H +-rich milieu that anticancer drugs meet once they get inside the tumor leads to their protonation and neutralization, therefore hindering their access into tumor cells. We have previously shown that proton pump inhibitors (PPI) may efficiently counterattack this tumor advantage leading to a consistent chemosensitization of tumors. In this study, we investigated the effects of PPI in chemosensitizing osteosarcoma. METHOD: MG-63 and Saos-2 cell lines were used as human osteosarcoma models. Cell proliferation after pretreatment with PPI and subsequent treatment with cisplatin was evaluated by using erythrosin B dye vital staining. Tumour growth was evaluated in xenograft treated with cisplatin after PPI pretreatment. Subsequently, a multi-centre historically controlled trial, was performed to evaluate the activity of a pre-treatment administration of PPIs as chemosensitizers during neoadjuvant chemotherapy based on methotrexate, cisplatin, and adriamycin. RESULTS: Preclinical experiments showed that PPI sensitize both human osteosarcoma cell lines and xenografts to cisplatin. A clinical study subsequently showed that pretreatment with PPI drug esomeprazole leads to an increase in the local effect of chemotherapy, as expressed by percentage of tumor necrosis. This was particularly evident in chondroblastic osteosarcoma, an histological subtype that normally shows a poor histological response. Notably, no significant increase in toxicity was recorded in PPI treated patients. CONCLUSION: This study provides the first evidence that PPI may be beneficially added to standard regimens in combination to conventional chemotherapy.