Alteration of glucogenic and glucogenic-ketogenic metabolites and nucleotide metabolism in MCF-7 breast cancer cells, treated with ribitol, ribose, and xylitol.

(A) All glucogenic amino acids levels were increased after ribitol treatment compared to ribose and xylitol. (B) Glucogenic and ketogenic amino acids such as tyrosine, phenylalanine and tryptophan levels were increased in ribitol treated cells compared to ribose and xylitol treatment. (C) Ribitol treatment increases the nucleotide synthesis by increasing the purines and pyrimidines synthesis, compared to ribose and xylitol treatment and Ribitol treatment significantly increased the level of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), one of the key intermediates of nucleotide synthesis. Significance denoted by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 within individual metabolite box plots, as determined by Welch’s two-sample t-Test.</p

Alteration in TCA Cycle metabolite or intermediates in MCF-7 cells treated with ribitol, ribose, and xylitol.

High level of pyruvate is associated with lower levers of citrate, isocitrate, indicating the conversion of pyruvate to acetyl-CoA is impeded. Increased level of alpha-KG to malate with ribitol treatment suggests an enhanced glutaminolysis and/or gluconeogenesis. Significant p>0.05 changes with ribitol indicated with increases in green and decreases in red in pathway schematic. Significance denoted by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 within individual metabolite box plots, as determined by Welch’s two-sample t-Test.</p

Schematic summary of metabolic and transcriptome alterations in MCF-7 breast cancer cells treated with ribitol.

Ribitol treated MCF 7 cells enhances various central carbon metabolic pathways like glycolysis, one carbon metabolism and nucleotide synthesis. Simultaneously, ribitol treatment decreases the ROS production, not entering into the TCA cycle (Acetyl Co-A not assayed in our study) and no fatty acid synthesis further. In addition, some of the TCA cycle intermediates were altered, this could be due to increased glutaminolysis. This may fuel the TCA cycle intermediates and their levels were altered in ribitol treatment. Metabolic pathways and the observed alterations in metabolites are depicted in black, while the associated enzymes found to be altered via transcriptomics are depicted as decreased expression (green) and increased expression (red) upon ribitol administration.</p

Heatmap of entire gene expression profiles of MCF-7 cells treated with ribitol, ribose, and xylitol by microarray analysis.

(A) Biological replicates labeled by treatment group across the X-axis and significantly altered genes on the Y-axis. (B) Expression is depicted by intensity, while differential expression increased or decreased is depicted by color. (FC ≥2, significant p>0.05 changes with ribitol indicated with increases in blue and decreases in red. Illustration of significantly altered transcripts by pentose sugars in breast cancer cells. (C) The genes listed represent the most significantly altered genes in each treatment when compared to the control and plays crucial roles in central carbon metabolism. Genes with alteration shared with ribitol, xylitol and ribose are indicated in dotted line squares and one gene shared between xylitol and ribose is indicated in solid line squares. The differential gene expression of these pentose sugars may be useful for identification of molecular signatures, analysis of networks or pathways and novel potential targets for treatment of cancer. Abbreviations: PCK1&2: Phosphoenol pyruvate carboxykinase 1&2; IDH: Isocitrate dehydrogenase; ACO2: Aconitase 2; GSR: Glutathione reductase; PHGDH: Phosphoglycerate dehydrogenase; ACLY: ATP citrate lyase. TALDO1: Trans aldolase 1; HK1: Hexokinase1; CS: Citrate synthase; PC: Pyruvate carboxylase; GSS: Glutathione synthetase; SDHD: Succinate dehydrogenase; LDH: Lactate dehydrogenase.</p

Matriglycan expression in MCF-7 cells treated with ribitol.

MCF-7 breast cancer cells were incubated for 72 hours with ribitol (10 mM) supplementation, with and without insulin in the base media, and analyzed by flow cytometry for detection of glycosylated α-dystroglycan (A) Representative histograms demonstrating the distribution and median X axis, fluorescence intensity; Y axis, number of cells. Red arrow indicates peak of highest MF, and black arrow indicate corresponding position in each histogram. (B) Mean fluorescence intensity of MCF7 cells after ribitol treatment. (C) Representative micrographs of IIH6 ICC staining for glycosylated matriglycan in untreated control and 10 mM ribitol supplementation with and without insulin in culture media. 2nd Ab-only control denotes staining with secondary antibody only, scale bar represents 200 μm. Metabolomic analysis of MCF7 cell lysates. (D) Hierarchical clustering analysis (HCA) heatmap of global metabolic profile alterations by pathway, amongst MCF-7 cells treated with 10 mM ribitol, 10 mM ribose, and 10 mM xylitol. (E) Comparison of statistically significant biochemical changes amongst metabolomic profiles of MCF-7 cells treated with ribitol, ribose, and xylitol. Cellular lysates compared by treatment to control, and between treatment groups. The two conditions compared make up the first row, with the total number of biochemicals reaching significance by two-sample t-Test p≤0.05 depicted in the second row, delineated by those increased or decreased in the third row. The count of biochemicals approaching significance, with two-sample t-Test 0.05</p

Combination Approach of YSA Peptide Anchored Docetaxel Stealth Liposomes with Oral Antifibrotic Agent for the Treatment of Lung Cancer

Therapeutic efficacy of nanocarriers can be amplified by active targeting and overcoming the extracellular matrix associated barriers of tumors. The aim of the present study was to investigate the effect of oral antifibrotic agent (telmisartan) on tumor uptake and anticancer efficacy of EphA2 receptor targeted liposomes. Docetaxel loaded PEGylated liposomes (DPL) functionalized with nickel chelated phospholipid were prepared using a modified hydration method. DPL were incubated with various concentrations of histidine tagged EphA2 receptor specific peptide (YSA) to optimize particle size, zeta potential, and percentage YSA binding. Cellular uptake studies using various endocytosis blockers revealed that a caveolae dependent pathway was the major route for internalization of YSA anchored liposomes of docetaxel (YDPL) in A549 lung cancer cell line. Hydrodynamic diameter and zeta potential of optimized YDPL were 157.3 ± 11.8 nm and −3.64 mV, respectively. Orthotopic lung tumor xenograft (A549) bearing athymic nude mice treated with oral telmisartan (5 mg/kg) for 2 days showed significantly (<i>p</i> < 0.05) higher uptake of YDPL in tumor tissues compared to healthy tissue. Average lung tumor weight of the YDPL + telmisartan treated group was 4.8- and 3.8-fold lower than that of the DPL and YDPL treated groups (<i>p</i> < 0.05). Substantially lower expression (<i>p</i> < 0.05) of EphA2 receptor protein, proliferating cell nuclear antigen (PCNA), MMP-9, and collagen 1A level with increased E-cadherin and TIMP-1 levels in immunohistochemistry and Western blot analysis of lung tumor samples of the combination group confirmed antifibrotic effect with enhanced anticancer activity. Active targeting and ECM remodeling synergistically contributed to anticancer efficacy of YDPL in orthotopic lung cancer

Characterization of BBR-SD and BA-SD formulations.

<p>A) DSC analysis of BBR free drug, Physical mixture and BBR-SD formulation. B) DSC analysis of BA free drug, Physical mixture and BA-SD formulation. Scanning electron microscopic analysis of C) BBR-SD and D) BA-SD formulation by using Zeiss FESEM.</p

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Real time-PCR analysis and Toxicity evaluation.

<p>A) Expression of p38, Phospho-JNK, Bax, Bcl-2, cleaved casapase-3&8, survivin and BAD in orthotopic lung tumor after treatment with BA free drug and BA-SD by real time PCR. B) Body weight changes during the treatment with BA free drug and BA-SD. C)Representative H&E stain based histopathological analysis of small intestine (jejunum) after treatment with BA free drug and BA-SD formulations. Each data point was represented as mean±sem (n = 6–8). **p<0.01 and ***p<0.001 Vs respective untreated control groups.</p

KRAS gene expression and protein levels in MCF-7, MDA MB-231, and T-47D breast cancer cells treated with ribitol.

A) KRAS expression in MCF-7 cell line by Real-Time qRT-PCR B) Relative KRAS expression in MDA MB-231 and T-47D breast cancer cell lines by Real-Time qRT-PCR C) KRAS protein expression by western blot of control, and ribitol-treated breast cancer cell lines MDA MB-231, MCF-7, and T-47D (D) Relative KRAS expression in xenograft MCF-7 tumors after ribitol treatment by Real-Time qRT-PCR.</p