Expression of RFC/SLC19A1 is Associated with Tumor Type in Bladder Cancer Patients

Urinary bladder cancer (UBC) ranks ninth in worldwide cancer. In Egypt, the pattern of bladder cancer is unique in that both the transitional and squamous cell types prevail. Despite much research on the topic, it is still difficult to predict tumor progression, optimal therapy and clinical outcome. The reduced folate carrier (RFC/SLC19A1) is the major transport system for folates in mammalian cells and tissues. RFC is also the primary means of cellular uptake for antifolate cancer chemotherapeutic drugs, however, membrane transport of antifolates by RFC is considered as limiting to antitumor activity. The purpose of this study was to compare the mRNA expression level of RFC/SLC19A1 in urothelial and non-urothelial variants of bladder carcinomas. Quantification of RFC mRNA in the mucosa of 41 untreated bladder cancer patients was performed using RT-qPCR. RFC mRNA steady-state levels were ∼9-fold higher (N = 39; P<0.0001) in bladder tumor specimens relative to normal bladder mRNA. RFC upregulation was strongly correlated with tumor type (urothelial vs. non-urothelial; p<0.05) where median RFC mRNA expression was significantly (p<0.05) higher in the urothelial (∼14-fold) compared to the non-urothelial (∼4-fold) variant. This may account for the variation in response to antifolate-containing regimens used in the treatment of either type. RFC mRNA levels were not associated with tumor grade (I, II and III) or stage (muscle-invasive vs. non-muscle invasive) implying that RFC cannot be used for prognostic purposes in bladder carcinomas and its increased expression is an early event in human bladder tumors pathogenesis. Further, RFC can be considered as a potential marker for predicting response to antifolate chemotherapy in urothelial carcinomas

Decitabine impact on the endocytosis regulator RhoA, the folate carriers RFC1 and FOLR1, and the glucose transporter GLUT4 in human tumors.

BackgroundIn 31 solid tumor patients treated with the demethylating agent decitabine, we performed tumor biopsies before and after the first cycle of decitabine and used immunohistochemistry (IHC) to assess whether decitabine increased expression of various membrane transporters. Resistance to chemotherapy may arise due to promoter methylation/downregulation of expression of transporters required for drug uptake, and decitabine can reverse resistance in vitro. The endocytosis regulator RhoA, the folate carriers FOLR1 and RFC1, and the glucose transporter GLUT4 were assessed.ResultsPre-decitabine RhoA was higher in patients who had received their last therapy &gt;3&nbsp;months previously than in patients with more recent prior therapy (P = 0.02), and varied inversely with global DNA methylation as assessed by LINE1 methylation (r = -0.58, P = 0.006). Tumor RhoA scores increased with decitabine (P = 0.03), and RFC1 also increased in patients with pre-decitabine scores ≤150 (P = 0.004). Change in LINE1 methylation with decitabine did not correlate significantly with change in IHC scores for any transporter assessed. We also assessed methylation of the RFC1 gene (alias SLC19A1). SLC19A1 methylation correlated with tumor LINE1 methylation (r = 0.45, P = 0.02). There was a small (statistically insignificant) decrease in SLC19A1 methylation with decitabine, and there was a trend towards change in SLC19A1 methylation with decitabine correlating with change in LINE1 methylation (r = 0.47, P &lt;0.15). While SLC19A1 methylation did not correlate with RFC1 scores, there was a trend towards an inverse correlation between change in SLC19A1 methylation and change in RFC1 expression (r = -0.45, P = 0.19).ConclusionsIn conclusion, after decitabine administration, there was increased expression of some (but not other) transporters that may play a role in chemotherapy uptake. Larger patient numbers will be needed to define the extent to which this increased expression is associated with changes in DNA methylation

Beyond reduction of atherosclerosis: PON2 provides apoptosis resistance and stabilizes tumor cells

Major contributors to atherosclerosis are oxidative damage and endoplasmic reticulum (ER) stress-induced apoptosis; both of which can be diminished by the anti-oxidative protein paraoxonase-2 (PON2). ER stress is also relevant to cancer and associated with anti-cancer treatment resistance. Hence, we addressed, for the first time, whether PON2 contributes to tumorigenesis and apoptotic escape. Intriguingly, we found that several human tumors upregulated PON2 and such overexpression provided resistance to different chemotherapeutics (imatinib, doxorubicine, staurosporine, or actinomycin) in cell culture models. This was reversed after PON2 knock-down. Remarkably, just deficiency of PON2 caused apoptosis of selective tumor cells per se, demonstrating a previously unanticipated oncogenic function. We found a dual mechanistic role. During ER stress, high PON2 levels lowered redox-triggered induction of pro-apoptotic CHOP particularly via the JNK pathway, which prevented mitochondrial cell death signaling. Apart from CHOP, PON2 also diminished intrinsic apoptosis as it prevented mitochondrial superoxide formation, cardiolipin peroxidation, cytochrome c release, and caspase activation. Ligand-stimulated apoptosis by TRAIL or TNFα remained unchanged. Finally, PON2 knock-down caused vast reactive oxygen species formation and stimulated JNK-triggered CHOP expression, but inhibition of JNK signaling did not prevent cell death, demonstrating the pleiotropic, dominating anti-oxidative effect of PON2. Therefore, targeting redox balance is powerful to induce selective tumor cell death and proposes PON2 as new putative anti-tumor candidate

Localized-Statistical Quantification of Human Serum Proteome Associated with Type 2 Diabetes

BACKGROUND: Recent advances in proteomics have shed light to discover serum proteins or peptides as biomarkers for tracking the progression of diabetes as well as understanding molecular mechanisms of the disease. RESULTS: In this work, human serum of non-diabetic and diabetic cohorts was analyzed by proteomic approach. To analyze total 1377 high-confident serum-proteins, we developed a computing strategy called localized statistics of protein abundance distribution (LSPAD) to calculate a significant bias of a particular protein-abundance between these two cohorts. As a result, 68 proteins were found significantly over-represented in the diabetic serum (p<0.01). In addition, a pathway-associated analysis was developed to obtain the overall pathway bias associated with type 2 diabetes, from which the significant over-representation of complement system associated with type 2 diabetes was uncovered. Moreover, an up-stream activator of complement pathway, ficolin-3, was observed over-represented in the serum of type 2 diabetic patients, which was further validated with statistic significance (p = 0.012) with more clinical samples. CONCLUSIONS: The developed LSPAD approach is well fit for analyzing proteomic data derived from biological complex systems such as plasma proteome. With LSPAD, we disclosed the comprehensive distribution of the proteins associated with diabetes in different abundance levels and the involvement of ficolin-related complement activation in diabetes

Antifolate resistance associated with loss of MRP1 expression and function in Chinese hamster ovary cells with markedly impaired export of folate and cholate

Export of folates from a Chinese hamster ovary PyrR100 cell line is markedly impaired, resulting in expansion of cellular folate pools and high-level antifolate resistance. We now report that MRP1 expression is absent in PyrR100 cells along with a marked decrease in MRP5 expression with 3-fold cross-resistance to thiopurines. PyrR100 and wild-type cells had comparable low levels of MRP2 expression; both lacked the breast cancer resistance protein. PyrR100 cells showed a 4-fold decrease in cholate (an MRP substrate) efflux with a 6-fold increase in cellular cholate accumulation compared with wild-type cells. Prostaglandin A1 increased cholate accumulation in wild-type cells to levels comparable with PyrR100 cells. Calcein (an MRP1 substrate) fluorescence increased 5-fold in PyrR100 cells; probenecid increased the intracellular calcein level in wild-type cells to that of PyrR100 cells. Consistent with the loss of MRP1 expression, PyrR100 cells showed modest collateral sensitivity to cholate, etoposide, doxorubicin, and vincristine. Transfection of MRP5 into PyrR100 cells did not alter sensitivity to pyrimethamine or MTX but restored sensitivity to mercaptopurines, indicating that decreased MRP5 expression did not play a role in antifolate resistance. Hence, although MRP-mediated anticancer drug resistance has been associated with gain of function (i.e., overexpression), this is the first report that loss of MRP1 efflux function can expand intracellular folate pools to result in acquired antifolate resistance. The data also suggest that MRP1, and possibly other MRPs that transport folates, can play a role in the maintenance of cellular folate homeostasis

Somatostatin Gene and Protein Expression in the Non-human Primate Central Extended Amygdala

Alterations in central extended amygdala (EAc) function have been linked to anxiety, depression, and anxious temperament (AT), the early-life risk to develop these disorders. The EAc is composed of the central nucleus of the amygdala (Ce), the bed nucleus of the stria terminalis (BST), and the sublenticular extended amygdala (SLEA). Using a non-human primate model of AT and multimodal neuroimaging, the Ce and the BST were identified as key AT-related regions. Both areas are primarily comprised of GABAergic neurons and the lateral Ce (CeL) and lateral BST (BSTL) have among the highest expression of neuropeptides in the brain. Somatostatin (SST) is of particular interest because mouse studies demonstrate that SST neurons, along with corticotropin-releasing factor (CRF) neurons, contribute to a threat-relevant EAc microcircuit. Although the distribution of CeL and BSTL SST neurons has been explored in rodents, this system is not well described in non-human primates. In situ hybridization demonstrated an anterior-posterior gradient of SST mRNA in the CeL but not the BSTL of non-human primates. Triple-labeling immunofluorescence staining revealed that SST protein-expressing cell bodies are a small proportion of the total CeL and BSTL neurons and have considerable co-labeling with CRF. The SLEA exhibited strong SST mRNA and protein expression, suggesting a role for SST in mediating information transfer between the CeL and BSTL. These data provide the foundation for mechanistic non-human primate studies focused on understanding EAc function in neuropsychiatric disorders