Ellagic Acid Resensitizes Gemcitabine-Resistant Bladder Cancer Cells by Inhibiting Epithelial-Mesenchymal Transition and Gemcitabine Transporters.

Gemcitabine (GCB) resistance is a major issue in bladder cancer chemoresistance, but its underlying mechanism has not been determined. Epithelial-mesenchymal transition (EMT) has been shown to be comprehensively involved in GCB resistance in several other cancer types, but the direct connection between EMT and GCB remains unclear. This study was designed to elucidate the mechanism of EMT-related GCB resistance in bladder cancer and identify a potential phytochemical to modulate drug sensitivity. The biological effects of ellagic acid (EA) or its combined effects with GCB were compared in GCB-resistant cells and the GCB-sensitive line in terms of cell viability, apoptosis, motility, and in vivo tumorigenicity. The molecular regulation of EMT-related GCB resistance was evaluated at both the mRNA and protein expression levels. Our results indicated that TGF-ß/Smad induced the overactivation of EMT in GCB-resistant cells and reduced the expression of GCB influx transporters (hCNT1 and hENT1). Moreover, ellagic acid (EA) inhibited the TGF-ß signaling pathway both in vitro and in vivo by reducing Smad2, Smad3, and Smad4 expression and thereby resensitized GCB sensitivity. In conclusion, our results demonstrate that TGF-ß/Smad-induced EMT contributes to GCB resistance in bladder cancer by reducing GCB influx and also elucidate the novel mechanisms of EA-mediated inhibition of TGF-ß/Smad-induced EMT to overcome GCB resistance. Our study warrants further investigation of EA as an effective therapeutic adjuvant agent for overcoming GCB resistance in bladder cancer.

Expression of the nucleoside transporters hENT1 (SLC29) and hCNT1 (SLC28) in pediatric acute myeloid leukemia.

Cellular uptake of clinically important deoxynucleoside analogs is mediated by nucleoside transporters including the human equilibrative nucleoside transporter 1 (hENT1) and the concentrative nucleoside transporter-1 (hCNT1). These transporters are responsible for influx of cytarabine and reduced hENT1 expression is a major resistance mechanism in acute myeloid leukemia. We determined hENT1 and hCNT1 protein expression by immunocytochemistry in 50 diagnostic pediatric acute myeloid leukemia patient samples. All samples expressed hENT1 [9/43 (21%) low; 26/43 (60%) medium and 8/43 (19%) high] and hCNT1 [2/42 (5%) low; 35/42 (83%) medium and 5/42 (12%) high] at the cell membrane and cytoplasm. Statistical analysis showed a non-significant relationship between survival and transporter expression and in vitro drug sensitivity. In conclusion, the nucleoside transporters hENT1 and hCNT1 are broadly expressed in pediatric acute myeloid leukemia at diagnosis.

A novel RNA variant of human concentrative nucleoside transporter 1 (hCNT1) that is a potential cancer biomarker.

BACKGROUND: The human concentrative nucleoside transporter 1 (hCNT1) a product of the SLC28A1 gene is one of the three concentrative nucleoside transporters, with a substrate specificity for physiological pyrimidine nucleosides. It has recently been implicated in tumor suppression. We have unraveled a splice variant RNA transcript that is overexpressed in some tumor tissues and some cancer cells. This study established  that observation. METHODS: We examined several clones of hCNT1 generated from RT-PCR of total RNA from human kidney tissue purchased from Ambion. The resulting cDNA clones were then sequenced, and a variant that retained intron 4, and skipped some exons fully or partly, specifically exons 5 and 13 were completely missed and only part of exon 6 was spliced. Tissue expression analysis by PCR indicated a similar distribution of expression of RNA of the splice variant hCNT1-IR as that of the dominant variant hCNT1, particularly in the small intestine, kidney and liver. Further, analysis of various tumor samples with PCR primers designed from this novel hCNT1 splice variant (hCNT1-IR) revealed interestingly that it is overexpressed in some cancer tissues relative to normal tissues, particularly kidney, liver and pancreatic cancers. CONCLUSION: We have identified a novel intron retaining and exon skipping splice variant of the hCNT1 nucleoside transporter, and designated it hCNT1-IR, which has a similar tissue expression distribution as the normal hCNT1 variant, but unlike the normal transcript, hCNT1-IR is overexpressed in some cancers and may serve as a potential cancer biomarker.

Deficiency of perforin and hCNT1, a novel inborn error of pyrimidine metabolism, associated with a rapidly developing lethal phenotype due to multi-organ failure.

Pyrimidine nucleotides are essential for a vast number of cellular processes and dysregulation of pyrimidine metabolism has been associated with a variety of clinical abnormalities. Inborn errors of pyrimidine metabolism affecting enzymes in the pyrimidine de novo and degradation pathway have been identified but no patients have been described with a deficiency in proteins affecting the cellular import of ribonucleosides. In this manuscript, we report the elucidation of the genetic basis of the observed uridine-cytidineuria in a patient presenting with fever, hepatosplenomegaly, persistent lactate acidosis, severely disturbed liver enzymes and ultimately multi-organ failure. Sequence analysis of genes encoding proteins directly involved in the metabolism of uridine and cytidine showed two variants c.1528C > T (p.R510C) and c.1682G > A (p.R561Q) in SLC28A1, encoding concentrative nucleotide transporter 1 (hCNT1). Functional analysis showed that these variants affected the three-dimensional structure of hCNT1, altered glycosylation and decreased the half-life of the mutant proteins which resulted in impaired transport activity. Co-transfection of both variants, mimicking the trans disposition of c.1528C > T (p.R510C) and c.1682G > A (p.R561Q) in the patient, significantly impaired hCNT1 biological function. Whole genome sequencing identified two pathogenic variants c.50delT; p.(Leu17Argfs*34) and c.853_855del; p.(Lys285del) in the PRF1 gene, indicating that our patient was also suffering from Familial Hemophagocytic Lymphohistiocytosis type 2. The identification of two co-existing monogenic defects might have resulted in a blended phenotype. Thus, the clinical presentation of isolated hCNT1 deficiency remains to be established.

Defective hCNT1 transport contributes to gemcitabine chemoresistance in ovarian cancer subtypes: overcoming transport defects using a nanoparticle approach.

Nucleoside analogs are used as chemotherapeutic options for the treatment of platinum-resistant ovarian cancers. Human concentrative nucleoside transporter 1 (hCNT1) is implicated in sensitizing solid tumors to nucleoside analogs although its role in determining drug efficacy in ovarian cancers remains unclear. Here we examined the functional expression of hCNT1 and compared its contributions toward gemcitabine efficacy in histological subtypes of ovarian cancer. Radioactivity analysis identified hCNT1-mediated (3)H-gemcitabine transport in ovarian cancer cells to be significantly reduced compared with that of normal ovarian surface epithelial cells. Biochemical and immunocytochemical analysis identified that unlike normal ovarian cells which expressed high levels of hCNT1 at the apical cell surface, the transporter was either diminished in expression and/or mislocalized in cell lines of various subtypes of ovarian cancer. Retroviral expression of hCNT1 selectively rescued gemcitabine transport in cell lines representing serous, teratocarcinoma, and endometrioid subtypes, but not clear cell carcinoma (CCC). In addition, exogenous hCNT1 predominantly accumulated in intracytoplasmic vesicles in CCC suggesting defective cellular trafficking of hCNT1 as a contributing factor to transport deficiency. Despite diminution of hCNT1 transport in the majority of ovarian cancers and apparent trafficking defects with CCC, the chemotherapeutic efficacy of gemcitabine was broadly enhanced in all subtypes when delivered via engineered nanoparticles (NPs). Additionally, by bypassing the transport requirement, the delivery of a gemcitabine-cisplatin combination in NP formulation increased their synergistic interactions. These findings uncover hCNT1 as a putative determinant for nucleoside analog chemoresistance in ovarian cancer and may help rationalize drug selection and delivery strategies for various histological subtypes of ovarian cancer.