High-Dose Vitamin C: Preclinical Evidence for Tailoring Treatment in Cancer Patients

Simple SummaryVitamin C is an indispensable micronutrient in the human diet due to the multiple functions it carries out in the body. Reports of clinical studies have indicated that, when administered at high dosage by the intravenous route, vitamin C may exert beneficial antitumor effects in patients with advanced stage cancers, including those refractory to previous treatment with chemotherapy. The aim of this article is to provide an overview of the current scientific evidence concerning the different mechanisms of action by which high-dose vitamin C may kill tumor cells. A special focus will be given to those mechanisms that provide the rationale basis for tailoring vitamin C treatment according to specific molecular alterations present in the tumor and for the selection of the most appropriate companion drugs.High-dose vitamin C has been proposed as a potential therapeutic approach for patients with advanced tumors who failed previous treatment with chemotherapy. Due to vitamin C complex pharmacokinetics, only intravenous administration allows reaching sufficiently high plasma concentrations required for most of the antitumor effects observed in preclinical studies (>0.250 mM). Moreover, vitamin C entry into cells is tightly regulated by SVCT and GLUT transporters, and is cell type-dependent. Importantly, besides its well-recognized pro-oxidant effects, vitamin C modulates TET enzymes promoting DNA demethylation and acts as cofactor of HIF hydroxylases, whose activity is required for HIF-1 alpha proteasomal degradation. Furthermore, at pharmacological concentrations lower than those required for its pro-oxidant activity (<1 mM), vitamin C in specific genetic contexts may alter the DNA damage response by increasing 5-hydroxymethylcytosine levels. These more recently described vitamin C mechanisms offer new treatment opportunities for tumors with specific molecular defects (e.g., HIF-1 alpha over-expression or TET2, IDH1/2, and WT1 alterations). Moreover, vitamin C action at DNA levels may provide the rationale basis for combination therapies with PARP inhibitors and hypomethylating agents. This review outlines the pharmacokinetic and pharmacodynamic properties of vitamin C to be taken into account in designing clinical studies that evaluate its potential use as anticancer agent

Vitamin C Deficiency in Patients With Acute Myeloid Leukemia

Vitamin C has been shown to play a significant role in suppressing progression of leukemia through epigenetic mechanisms. We aimed to study the role of vitamin C in acute myeloid leukemia (AML) biology and clinical course. To this purpose, the plasma levels of vitamin C at diagnosis in 62 patients with AML (including 5 cases with acute promyelocytic leukemia, APL),7 with myelodysplastic syndrome (MDS), and in 15 healthy donors (HDs) were studied. As controls, vitamins A and E levels were analysed. Expression of the main vitamin C transporters and of the TET2 enzyme were investigated by a specific RQ-PCR while cytoplasmic vitamin C concentration and its uptake were studied in mononuclear cells (MNCs), lymphocytes and blast cells purified from AML samples, and MNCs isolated from HDs. There were no significant differences in vitamin A and E serum levels between patients and HDs. Conversely, vitamin C concentration was significantly lower in AML as compared to HDs (p<0.0001), inversely correlated with peripheral blast-counts (p=0.029), significantly increased at the time of complete remission (CR) (p=0.04) and further decreased in resistant disease (p=0.002). Expression of the main vitamin C transporters SLC23A2, SLC2A1 and SLC2A3 was also significantly reduced in AML compared to HDs. In this line, cytoplasmic vitamin C levels were also significantly lower in AML-MNCs versus HDs, and in sorted blasts compared to normal lymphocytes in individual patients. No association was found between vitamin C plasma levels and the mutation profile of AML patients, as well as when considering cytogenetics or 2017 ELN risk stratification groups. Finally, vitamin C levels did not play a predictive role for overall or relapse-free survival. In conclusion, our study shows that vitamin C levels are significantly decreased in patients with AML at the time of initial diagnosis, further decrease during disease progression and return to normal upon achievement of CR. Correspondingly, low intracellular levels may mirror increased vitamin C metabolic consumption in proliferating AML cells

Acute Delta Hepatitis in Italy spanning three decades (1991–2019): Evidence for the effectiveness of the hepatitis B vaccination campaign

Updated incidence data of acute Delta virus hepatitis (HDV) are lacking worldwide. Our aim was to evaluate incidence of and risk factors for acute HDV in Italy after the introduction of the compulsory vaccination against hepatitis B virus (HBV) in 1991. Data were obtained from the National Surveillance System of acute viral hepatitis (SEIEVA). Independent predictors of HDV were assessed by logistic-regression analysis. The incidence of acute HDV per 1-million population declined from 3.2 cases in 1987 to 0.04 in 2019, parallel to that of acute HBV per 100,000 from 10.0 to 0.39 cases during the same period. The median age of cases increased from 27 years in the decade 1991-1999 to 44 years in the decade 2010-2019 (p < .001). Over the same period, the male/female ratio decreased from 3.8 to 2.1, the proportion of coinfections increased from 55% to 75% (p = .003) and that of HBsAg positive acute hepatitis tested for by IgM anti-HDV linearly decreased from 50.1% to 34.1% (p < .001). People born abroad accounted for 24.6% of cases in 2004-2010 and 32.1% in 2011-2019. In the period 2010-2019, risky sexual behaviour (O.R. 4.2; 95%CI: 1.4-12.8) was the sole independent predictor of acute HDV; conversely intravenous drug use was no longer associated (O.R. 1.25; 95%CI: 0.15-10.22) with this. In conclusion, HBV vaccination was an effective measure to control acute HDV. Intravenous drug use is no longer an efficient mode of HDV spread. Testing for IgM-anti HDV is a grey area requiring alert. Acute HDV in foreigners should be monitored in the years to come

Autophagy inhibition enhances Natural Killer cell- based therapy in high-risk Medulloblastoma

Medulloblastoma (MB) is the most common pediatric brain tumor with Group3 (G3) subtype characterized by poor prognosis and therapy relapse. Autophagy is a self-degradative process increased in G3 MB stem cells (MBSCs) to contribute stemness and survival (Nazio et al., 2021). A promising therapeutic value in the treatment of high-risk MB is represented by Natural Killer cells (NKs)-based immunotherapy but it requires complementary approaches that break im- mune tolerance. To date, the role of autophagy-mediated mechanisms in regulating tumour heterogeneity and immune cells infiltration capability in the context of brain TME remains un- known as well as the role of autophagy in NK/CAR-NK mediated therapy is completely unex- plored. Herein, we are investigating the role of autophagy inhibition as a druggable mechanism to increase NK recognition and killing against MB G3. By flow cytometry analysis, we found low levels of NK-related activating ligands (ULBPs, CD155, CD112, MICA/B) in G3 cell lines and MBSCs derived by MB G3 patients compared to SHH subgroup. Intriguingly, genetic and pharmacologic inhibition of autophagy is able to increase NK-related ligand expression on cell surface of MB G3 cells. Additionally, by means of 2D and 3D models of MB G3 cells, we found that autophagy inhibition increases NK degranulation and MB G3 sensitiveness to NK-mediated cytotoxicity. Im- munotherapeutic strategies aimed at restoring and increasing the cytotoxic activity of NK cells in solid tumors, including the adoptive transfer of NK cells, are currently employed in preclinical and clinical studies. New strategies are necessary to make NK cells more resistant to the meta- bolically restrictive TME as well as to immunosuppressive molecules generated by the tumor. The completion of our project would be helpful to design a novel therapeutic approach for chil- dren with high-risk MB, aiming to a higher clinical response rate coupled with less toxicity as compared to conventional therapies

Fluorescent molecular rotors as sensors for the detection of thymidine phosphorylase

[Display omitted] Three new fluorescent molecular rotors were synthesized with the aim of using them as sensors to dose thymidine phosphorylase, one of the target enzymes of 5-fluorouracil, a potent chemotherapic drug largely used in the treatment of many solid tumors, that acts by hindering the metabolism of pyrimidines. 5-Fluorouracil has a very narrowtherapeutic window, in fact, its optimal dosage is strictly related to the level of its target enzymes that vary significantly among patients, and it would be of the utmost importance to have an easy and fast method to detect and quantify them. The three molecular rotors developed as TP sensors differ in the length of the alkylic spacer joining the ligand unit, a thymine moiety, and the fluorescent molecular rotor, a [4-(1-dimethylamino)phenyl]-pyridinium bromide. Their ability to trigger an optical signal upon the interaction with thymidine phosphorylase was investigated by fluorescent measurements

Synthesis, characterization and inclusion into liposomes of a new cationic pyrenyl amphiphile

The aggregation properties of a new cationic fluorescent amphiphile tagged on the hydrophobic tail with a pyrene moiety and bearing two hydroxyethyl functionalities on the polar headgroup were investigated by fluorescence experiments as pure components or in mixed liposomes containing an unsaturated phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine, at different molar ratios. The obtained results put in evidence that the conformation and the miscibility of the lipids in the aggregates strongly influence the excimer/monomer ratio. Mixed monolayers at the same composition were investigated by Langmuir compression isotherms to deepen the understanding of lipid organization and miscibility, both in the polar and in the hydrophobic regions. The presence of two hydroxyethyl functionalities on the polar headgroup of the newly synthesized amphiphile exerts a shielding effect of the charge of the amphiphile increasing the compressibility of lipid components in contrast with the disturbing effect of the unsaturated acyl chains of the phospholipid

Fluorescent lipid based sensor for the detection of thymidine phosphorylase as tumor biomarker

5-Fluorouracil (5-FU) is a chemotherapic drug widely employed to treat a wide range of solid tumors. Unfortunately, it has a narrow therapeutic window and the level of its target enzymes in biological fluids of patients can vary considerably. On these premises, a new fluorescent lipid based sensor for the detection of thymidine phosphorylase, one of the target enzymes of 5-FU, was developed, to optimize patient treatment. Both cationic and anionic fluorescent liposomes containing both an amphiphile tailtagged with a pyrene residue and a 5-FU derivative were investigated. The effect ofthe presence of a bulky quencher (the bromine atom) covalently linked to the end of the alkyl chain of the anionic component on the emission signal was also evaluated. The interaction of liposomes with the target enzyme induces the occurrence of a fluorescent signal, at an extent that depends on the formulation, due to the variation of the excimer/monomer ratio. In particular, a promising specific result was obtained upon the interaction of the target enzyme with liposomes formulated with DOPC, the cationic fluorescent surfactant, the 5-FU derivative and 11-bromoundecaonic acid at 5/1/1/3 molar ratio. Langmuir compression isotherms allowed clarifying the influence of lipid organization on the response of the sensor

Exploiting autophagy balance in T and NK cells as a new strategy to implement adoptive cell therapies

Abstract Autophagy is an essential cellular homeostasis pathway initiated by multiple stimuli ranging from nutrient deprivation to viral infection, playing a key role in human health and disease. At present, a growing number of evidence suggests a role of autophagy as a primitive innate immune form of defense for eukaryotic cells, interacting with components of innate immune signaling pathways and regulating thymic selection, antigen presentation, cytokine production and T/NK cell homeostasis. In cancer, autophagy is intimately involved in the immunological control of tumor progression and response to therapy. However, very little is known about the role and impact of autophagy in T and NK cells, the main players in the active fight against infections and tumors. Important questions are emerging: what role does autophagy play on T/NK cells? Could its modulation lead to any advantages? Could specific targeting of autophagy on tumor cells (blocking) and T/NK cells (activation) be a new intervention strategy? In this review, we debate preclinical studies that have identified autophagy as a key regulator of immune responses by modulating the functions of different immune cells and discuss the redundancy or diversity among the subpopulations of both T and NK cells in physiologic context and in cancer

Kinetics and mechanistic study of competitive inhibition of thymidinephosphorylase by 5-fluoruracil derivatives

In a previous investigation, cationic liposomes formulated with new 5-FU derivatives, differing for the length of the polyoxyethylenic spacer that links the N3position of 5-FU to an alkyl chain of 12 carbon atoms, showed a higher cytotoxicity compared to free 5-FU, the cytotoxic effect being directly related to the length of the spacer. To better understand the correlation of the spacer length with toxicity, we carried out initial rate studies to determine inhibition, equilibrium and kinetic constants (KI, KM, kcat), and get inside inhibition activity of the 5-FU derivatives and their mechanism of action, a crucial information to design structural variations for improving the anticancer activity. The experimental investigation was supported by docking simulations based on the X-ray structure of thymidine phosphorylase (TP) from Escherichia coli complexed with 3'-azido-2'-fluoro-dideoxyuridin. Theoretical and experimental results showed that all the derivatives exert the same inhibition activity of 5-FU either as monomer and when embedded in lipid bilayer