High Doses of Ascorbate Kill Y79 Retinoblastoma Cells In vitro

Objectives: To tests the sensitivity of Y79 retinoblastoma cell lines to high doses of ascorbate, in vitro, and compare its effects with those of some chemotherapeutic agents routinely employed in the treatment of retinoblastoma. Methods: Y79 retinoblastoma cells have been exposed to increasing doses of either sodium ascorbate (SA) or Melphalan (MEL), to define a dose-response curve around the peak plasma concentrations reached by both chemicals when administered according to the existing therapeutic procedures and protocols. The assessment of cell number and viability was performed, before and after exposure, with both the manual (Trypan Blue Exclusion Test) and automated (flow cytometry) methods. Fluorescence microscopy and direct observation of cells in culture, with inverted microscope, were also performed. Results: Y79 cells are highly sensitive to the cytotoxic effect of SA, with cell viability reduced of over 90% in some experiments. As reported in the literature, this effect is directly cytotoxic and most probably mediated by acute oxidative stress on different cellular components. The same does not apply to Melphalan which, at the doses commonly used for therapeutic purposes, did not show any significant effect on cell viability, in vitro. Conclusion: To our knowledge, this is the first report showing that high doses of SA can actively kill retinoblastoma cells in vitro. While it is not surprising for SA, to show direct cytotoxic effect on tumor cells, the data reported herein represent the first evidence in favor of the possible clinical use of high doses of intravenous SA, to treat children affected by retinoblastoma. Given the many advantages of SA over the chemotherapeutic agents commonly employed to treat cancer (including its almost total absence of toxic or side effects, and its exclusive specificity for cancer cells), it is reasonable to assume, from the data reported herein, that the high doses of intravenous ascorbate, have the potential to represent a real revolution in the treatment of retinoblastoma

Demographic characteristics of the study population.

<p>IQR, interquartile range; HE, heterosexual; MSM, men-who-have-sex-with-men; IDU, injection drug user; ND, not determined; IU, international units.</p

The graph reports the distribution of FPR values of all the V3 variants detected by UDPS in each patient according to FPR ranges at population V3 sequencing.

<p>The relative dimension of green and red dots represents the prevalence of R5 and X4 species detected by UDPS. Yellow dots represent the FPR determined by population sequencing and letters within dots indicate the phenotypic tropism determined by ESTA (R =  pure CCR5 tropism, X = pure CXCR4 tropism, D = dual/mixed tropism. For blank yellow dots, ESTA result was not available. A FPR of 5.75 has been used as cut-off to infer HIV-1 co-receptor usage of V3 sequences obtained by both V3 population and ultra-deep sequencing.</p

The graphs report the proportion of R5 (A) and X4 (B) variants per patient according to the values of FPR at population V3 sequencing.

<p>Distribution of R5 and X4 variants in relationship to the False Positive Rate (FPR) detected by population V3 sequencing. The graphs report the proportion of R5 (A) and X4 (B) variants per patient according to the values of FPR at population V3 sequencing. P-values were calculated by Spearman test. A FPR of 5.75 has been used as cut-off to infer HIV-1 co-receptor usage.</p

UDPS species prevalence according to G2P FPR at population V3 genotyping.

a<p>The column reports the range of prevalence for CXCR4-using or CCR5-using strains determined by UDPS in patients stratified according to the FPR values obtained by V3 population sequencing.</p>b<p>The ranges are referred to the intra-patient prevalence of X4- and R5-species by UDPS.</p>c<p>The intra-patient prevalence of X4-variants is 99.1% and 100%, respectively.</p><p>Abbreviations: UDPS, ultra-deep sequencing; G2P, Geno2Pheno; FPR, false positive rate.</p

Quasispecies heterogeneity.

<p>The box plots represent the diversity of amino acid sequences (A), and Shannon entropy (B) among patients with X4 species detected by UDPS at a prevalence lower or higher than 1%. P-values were calculated by Mann-Whitney Test.</p

Performance of genotypic tropism testing in clinical practice using the enhanced sensitivity version of Trofile as reference assay: results from the OSCAR Study Group

The goal of the OSCAR programme is to evaluate the performances of genotypic HIV-1 tropism testing in clinical practice using the enhanced sensitivity version of Trofile (ESTA) as reference-assay