The prognostic and predictive role of class III ß-Tubulin and hENT1 expression in patients with advanced pancreatic ductal adenocarcinoma.

BACKGROUND: FOLFIRINOX and gemcitabine-nabpaclitaxel (GnP) are standard first-line treatment regimens for advanced pancreatic ductal adenocarcinoma (PDAC). However, currently, there is a lack of predictive biomarkers to aid in the treatment selection. We aimed to explore the prognostic and predictive value of class III ß-Tubulin (TUBB3) and human equilibrative nucleoside transporter 1 (hENT1) expression, which have previously been shown to be associated with taxane and gemcitabine resistance in advanced PDAC. METHODS: We conducted a retrospective analysis of 106 patients with advanced PDAC treated with GnP and/or FOLFIRINOX at our institution. TUBB3 and hENT1 immunohistochemical staining was performed on tumor specimens and subsequently evaluated based on the intensity and percentage of expression. RESULTS: In patients who received the GnP regimen, a high combined score (TUBB3low/hENT1high) was associated with a higher DCR and longer PFS compared to those with intermediate (TUBB3high/hENT1high or TUBB3low/hENT1low) and low score (TUBB3high/hENT1low). In the multivariate analysis, a high combined score was an independent predictor of higher DCR (OR:11.96; 95 % CI:2.61-54.82; p = 0.001) and longer PFS (HR:0.33; 95%CI:0.18-0.60; p < 0.001). However, there was no difference in response rates or PFS based on TUBB3 and hENT1 expression among patients receiving the FOLFIRINOX regimen. CONCLUSION: Our findings indicate that tumor TUBB3 and hENT1 expression may predict the efficacy of the GnP regimen, and low TUBB3 and high hENT1 expression (TUBB3low/hENT1high) are associated with a higher DCR and longer PFS in patients treated with GnP. Evaluating TUBB3 and hENT1 jointly can identify the patients most (as well as least) likely to benefit from GnP chemotherapy.

Kinetic profiling of novel spirobenzo-oxazinepiperidinone derivatives as equilibrative nucleoside transporter 1 inhibitors.

Evaluation of kinetic parameters of drug-target binding, kon, koff, and residence time (RT), in addition to the traditional in vitro parameter of affinity is receiving increasing attention in the early stages of drug discovery. Target binding kinetics emerges as a meaningful concept for the evaluation of a ligand's duration of action and more generally drug efficacy and safety. We report the biological evaluation of a novel series of spirobenzo-oxazinepiperidinone derivatives as inhibitors of the human equilibrative nucleoside transporter 1 (hENT1, SLC29A1). The compounds were evaluated in radioligand binding experiments, i.e., displacement, competition association, and washout assays, to evaluate their affinity and binding kinetic parameters. We also linked these pharmacological parameters to the compounds' chemical characteristics, and learned that separate moieties of the molecules governed target affinity and binding kinetics. Among the 29 compounds tested, 28 stood out with high affinity and a long residence time of 87 min. These findings reveal the importance of supplementing affinity data with binding kinetics at transport proteins such as hENT1.

hENT1's role in adjuvant intra-arterial gemcitabine-based chemotherapy for resectable pancreatic cancer patients.

BACKGROUND: We aimed to verify the role of hENT1 as a prognostic predictor for patients with resectable pancreatic ductal adenocarcinoma (PDAC) who underwent radical resection followed by intra-arterial infusion of gemcitabine-based regimen. METHODS: We collected surgical samples from 102 patients with resectable PDAC who received radical resection followed by intra-arterial infusion of gemcitabine-based regimen. The hENT1 expression with the help of immunohistochemistry was conducted using formalin-fixed and paraffin embedded tissues. The Kaplan-Meier analyses and Cox regression were used to evaluate the mortality hazard associated with the discrepancy between strong and weak of hENT1 expression. Patients' clinical and pathological characteristics were compared between the two groups, then the role of hENT1 as a prognostic predictor was further explored. RESULTS: A total of 102 patients were included to assess the hENT1 expression. 50 patients were classified into high hENT1 expression group, the other 52 patients were attributed into low hENT1 expression group. High hENT1 expression was related to a significantly improved overall survival (OS) (p = 0.014) and disease-free survival (DFS) (p = 0.004). Both univariate (p = 0.001) and multivariate analyses (p < 0.001) indicated that high hENT1 expression was related to a decreased mortality. CONCLUSIONS: High expression of hENT1 is positive prognostic factor for adjuvant intra-arterial gemcitabine-based chemotherapy in resectable PDAC.

Combination treatment with hENT1 and miR-143 reverses gemcitabine resistance in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer and is susceptible to develop gemcitabine (GEM) resistance. Decreased expression of human equilibrative nucleoside transporter 1 (hENT1) accompanied by compensatory increase of glycolysis is strongly associated with GEM resistance in TNBC. In this study, we investigated the treatment feasibility of combined hENT1 upregulation and miR-143-mediated inhibition of glycolysis for reversing GEM resistance in TNBC. METHODS: Experiments were performed in vitro and in vivo to compare the efficacy of GEM therapies. In this study, we established stable drug-resistant cell line, GEM-R cells, from parental cells (MDA-MB-231) through exposure to GEM following a stepwise incremental dosing strategy. Then GEM-R cells were transfected by lentiviral plasmids and GEM-R cells overexpressing hENT1 (GEM-R-hENT1) were established. The viability and apoptosis of wild-type (MDA-MB-231), GEM-R, and GEM-R-hENT1 cells treated with GEM or GEM + miR-143 were analyzed by CCK8 assay and flow cytometry. The RNA expression and protein expression were measured by RT-PCR and western blotting respectively. GEM uptake was determined by multiple reaction monitoring (MRM) analysis. Glycolysis was measured by glucose assay and 18F-FDG uptake. The antitumor effect was assessed in vivo in a tumor xenograft model by evaluating toxicity, tumor volume, and maximum standardized uptake value in 18F-FDG PET. Immunohistochemistry and fluorescence photography were taken in tumor samples. Pairwise comparisons were performed using Student's t-test. RESULTS: Our results represented that overexpression of hENT1 reversed GEM resistance in GEM-R cells by showing lower IC50 and higher rate of apoptosis. MiR-143 suppressed glycolysis in GEM-R cells and enhanced the effect of reversing GEM resistance in GEM-R-hENT1 cells. The therapeutic efficacy was validated using a xenograft mouse model. Combination treatment decreased tumor growth rate and maximum standardized uptake value in 18F-FDG PET more effectively. CONCLUSIONS: Combined therapy of exogenous upregulation of hENT1 expression and miR-143 mimic administration was effective in reversing GEM resistance, providing a promising strategy for treating GEM-resistant TNBC.

Oligomerization of equilibrative nucleoside transporters: a novel regulatory and functional mechanism involving PKC and PP1.

Equilibrative nucleoside transporters (ENTs) translocate nucleosides and nucleobases across plasma membranes, as well as a variety of anti-cancer, -viral, and -parasite nucleoside analogs. They are also key members of the purinome complex and regulate the protective and anti-inflammatory effects of adenosine. Despite their important role, little is known about the mechanisms involved in their regulation. We conducted membrane yeast 2-hybrid and coimmunoprecipitation studies and identified, for the first time to our knowledge, the existence of protein-protein interactions between human ENT1 and ENT2 (hENT1 and hENT2) proteins in human cells and the formation of hetero- and homo-oligomers at the plasma membrane and the submembrane region. The use of NanoLuc Binary Technology allowed us to analyze changes in the oligomeric status of hENT1 and hENT2 and how they rapidly modify the uptake profile for nucleosides and nucleobases and allow cells to respond promptly to external signals or changes in the extracellular environment. These changes in hENTs oligomerization are triggered by PKC activation and subsequent action of protein phosphatase 1.-Grañe-Boladeras, N., Williams, D., Tarmakova, Z., Stevanovic, K., Villani, L. A., Mehrabi, P., Siu, K. W. M., Pastor-Anglada, M., Coe, I. R. Oligomerization of equilibrative nucleoside transporters: a novel regulatory and functional mechanism involving PKC and PP1.

Physicochemical and in vitro cytotoxicity studies of inclusion complex between gemcitabine and cucurbit[7]uril host.

Gemcitabine, a cytostatic drug from the pyrimidine antimetabolite group, exhibits limited storage stability and numerous side effects during therapy. One of the strategies to improve the effectiveness of therapy with such drugs is the use of supramolecular nano-containers, including dendrimers and macrocyclic compounds. The ability of gemcitabine to attach a proton in an aqueous environment necessitates the search for a carrier that is well-tolerated by an organism and capable of supramolecular binding of a ligand (drug) in a cationic form. In the current study a promising strategy was tested for using cucurbituril Q7 to bind gemcitabine cations for its efficient intracellular delivery on three selected cancer cell lines (MOLT4, THP-1 and U937). Based on physicochemical studies (equilibrium dialysis, UV and 1H NMR titrations, DOSY 1H NMR measurements, DSC calorimetry) and cytotoxicity tests on cells with a free and blocked hENT1 transporter, the conclusion was drawn about the binding and penetration of the cucurbituril-drug complex into cancer cells.

Role of cysteine 416 in N-ethylmaleimide sensitivity of human equilibrative nucleoside transporter 1 (hENT1).

Human equilibrative nucleoside transporter 1 (hENT1), the first identified member of the ENT family of integral membrane proteins, is the primary mechanism for cellular uptake of physiologic nucleosides and many antineoplastic and antiviral nucleoside drugs. hENT1, which is potently inhibited by nitrobenzylthioinosine (NBMPR), possesses 11 transmembrane helical domains with an intracellular N-terminus and an extracellular C-terminus. As a protein with 10 endogenous cysteine residues, it is sensitive to inhibition by the membrane permeable sulfhydryl-reactive reagent N-ethylmaleimide (NEM) but is unaffected by the membrane impermeable sulfhydryl-reactive reagent p-chloromercuriphenyl sulfonate. To identify the residue(s) involved in NEM inhibition, we created a cysteine-less version of hENT1 (hENT1C-), with all 10 endogenous cysteine residues mutated to serine, and showed that it displays wild-type uridine transport and NBMPR-binding characteristics when produced in the Xenopus oocyte heterologous expression system, indicating that endogenous cysteine residues are not essential for hENT1 function. We then tested NEM sensitivity of recombinant wild-type hENT1, hENT1 mutants C1S to C10S (single cysteine residues replaced by serine), hENT1C- (all cysteine residues replaced by serine), and hENT1C- mutants S1C to S10C (single serine residues converted back to cysteine). Mutants C9S (C416S/hENT1) and S9C (S416C/hENT1C-) were insensitive and sensitive, respectively, to inhibition by NEM, identifying Cys416 as the endofacial cysteine residue in hENT1 responsible for NEM inhibition. Kinetic experiments suggested that NEM modification of Cys416, which is located at the inner extremity of TM10, results in the inhibition of hENT1 uridine transport and NBMPR binding by constraining the protein in its inward-facing conformation.

Human equilibrative nucleoside transporter 1 testing in pancreatic ductal adenocarcinoma: a comparison between murine and rabbit antibodies.

AIMS: The human equilibrative nucleoside transporter 1 (hENT1) expression level in pancreatic ductal adenocarcinoma (PDAC) may predict survival in gemcitabine-treated patients after resection. These results have been obtained with a murine anti-hENT1 antibody (10D7G2) that is not commercially available. Another antibody, which is rabbit-derived (SP120), appears to have no predictive value in local, advanced or metastatic PDAC. We aimed to study whether the two antibodies are equivalent. METHODS AND RESULTS: We compared hENT1 expression with both antibodies in resected PDAC. The results were correlated with overall survival (OS) following gemcitabine treatment. Tissues from two sets of patients (n = 147 each) were stained with SP120 by the use of different equipment, with an amplification technique being used for set 2. The rate of 'hENT1 high' cases was lower with SP120 (set 1, 7% versus 48%; set 2, 11% versus 38%). With the amplification technique, the rate of hENT1 high cases was globally similar between both antibodies. However, concordance between the antibodies was found in only 50% of cases. High hENT1 expression was predictive of OS only with 10D7G2 (hazard ratio 0.49; 95% confidence interval 0.24-0.98; P = 0.045). CONCLUSIONS: The two antibodies are not equivalent. Further prospective studies seem to be warranted before hENT1 testing for PDAC is used in daily practise.

A phase II, open-label, multicenter study to evaluate the antitumor efficacy of CO-1.01 as second-line therapy for gemcitabine-refractory patients with stage IV pancreatic adenocarcinoma and negative tumor hENT1 expression.

BACKGROUND: Nucleotide transporters such as human equilibrative nucleoside transporter-1 (hENT1) play a major role in transporting gemcitabine into cells. CO-1.01 (gemcitabine-5'-elaidate) is a novel cytotoxic agent consisting of a fatty acid derivative of gemcitabine, which is transported intracellularly independent of hENT1. CO-1.01 was postulated to have efficacy as a second-line treatment in gemcitabine-refractory pancreatic adenocarcinoma in patients with negative tumor hENT1 expression. METHODS: Eligibility criteria included patients with either a newly procured or archival biopsy tumor confirming the absence of hENT1 and either gemcitabine-refractory metastatic pancreas adenocarcinoma or with progression of disease following resection during or within 3 months of adjuvant gemcitabine therapy. Patients were treated with intravenous infusion of CO-1.01 dosed at 1250 mg/m(2) on Days 1, 8, and 15 of a 4-week cycle. The primary end point was disease control rate (DCR). RESULTS: Nineteen patients were enrolled of which 18 patients were evaluable for efficacy assessment. Thirteen patients (68%) had liver metastases, 6 (32%) had lymph node metastases, and 10 (53%) had lung metastases. Two of 18 patients (11%) achieved disease control. The median survival time was 4.3 (95% CI 2.1-8.1) months. All patients experienced at least one treatment-related adverse event with the majority of events being mild or moderate. CONCLUSION: This study did not meet its primary endpoint and no efficacy signal was identified for CO-1.01 in treating progressive metastatic pancreas adenocarcinoma.

Dilazep analogues for the study of equilibrative nucleoside transporters 1 and 2 (ENT1 and ENT2).

As ENT inhibitors including dilazep have shown efficacy improving oHSV1 targeted oncolytic cancer therapy, a series of dilazep analogues was synthesized and biologically evaluated to examine both ENT1 and ENT2 inhibition. The central diamine core, alkyl chains, ester linkage and substituents on the phenyl ring were all varied. Compounds were screened against ENT1 and ENT2 using a radio-ligand cell-based assay. Dilazep and analogues with minor structural changes are potent and selective ENT1 inhibitors. No selective ENT2 inhibitors were found, although some analogues were more potent against ENT2 than the parent dilazep.

Pancreatic cancer with liver metastasis maintaining complete response with gemcitabine monotherapy: A case report.

Pancreatic ductal adenocarcinoma (PDAC) is associated with a poor prognosis, and it has a recurrence rate of >70%, even in resectable cases. The treatment strategy for recurrent PDAC involves systemic chemotherapy, with gemcitabine (GEM) monotherapy historically serving as the standard of care. The present study describes the case of a patient with PDAC and postoperative liver metastases that maintained clinical complete remission (cCR) for >7 years following GEM monotherapy. A 63-year-old woman with upper abdominal pain was diagnosed with resectable PDAC and underwent pancreaticoduodenectomy. The patient was treated with GEM + S-1 as adjuvant chemotherapy for 6 months. Multiple liver metastases were detected 15 months post-operation and the patient was administered GEM alone. After 12 cycles, computed tomography showed cCR and GEM monotherapy was discontinued after 15 cycles. The patient has had no signs or symptoms of recurrence >7 years after the first recurrence. In addition, the present study analyzed PDAC resection specimens from four patients, including this case, to determine the expression levels of hENT1 protein in the tumor tissues. hENT1 is a transmembrane protein that acts as a nucleoside transporter and is a major mediator of GEM uptake into human cells. In the present case, hENT1 staining exhibited low frequency and weak positivity in the central region, whereas a strong positive reaction was observed in nearly all cell membranes at the invasive front of the cancer. The location, intensity, and frequency of hENT1 staining varied among cases. In conclusion, the efficacy of GEM may be predicted prior to treatment by evaluating hENT1 expression.

Human Equilibrative Nucleoside Transporter 1: Novel Biomarker and Prognostic Indicator for Patients with Gemcitabine-Treated Pancreatic Cancer.

Aim: This article aimed to find appropriate pancreatic cancer (PC) patients to treat with Gemcitabine with better survival outcomes by detecting hENT1 levels. Methods: We collected surgical pathological tissues from PC patients who received radical surgery in our hospital from September 2004 to December 2014. A total of 375 PC tissues and paired adjacent nontumor tissues were employed for the construction of 4 tissue microarrays (TMAs). The quality of the 4 TMAs was examined by HE staining. We performed immunohistochemistry analysis to evaluate hENT1 expression in the TMAs. Moreover, we detected hENT1 expression level and proved the role of hENT1 in cell proliferation, drug resistance, migration and invasion in vivo and vitro. Results: The results indicated that low hENT1 expression indicated a significantly poor outcome in PC patients, including shortened DFS (21.6±2.8 months versus 36.9±4.0 months, p<0.001) and OS (33.6±3.9 versus 39.6±3.9, p=0.004). Meanwhile, patients in stage I/II of TNM stage had a longer OS (40.2±3.4 versus 15.4±1.7, p=0.002) and DFS (31.0±3.1 versus 12.4±1.9, p=0.016) than patients in stage III/IV. Patients in M0 stage had a longer OS (39.7±3.4 versus 16.2±1.9, p=0.026) and DFS(30.7±3.0 versus 11.8±2.2, p=0.031) than patients in M1 stage, and patients with tumors not invading the capsule had a better DFS than those with tumor invasion into the capsule (30.8±3.0 versus 12.6±2.3, p=0.053). Patients with preoperative CA19-9 values ≤467 U/mL have longer DFS than that of patients who had preoperative CA19-9 values >467 U/mL (37.9±4.1 versus 22.9±4.0, p=0.04). In the subgroup analysis, a high hENT1 expression level was related to a longer OS(39.4±4.0 versus 31.5±3.9, p=0.001) and DFS(35.7±4.0 versus 20.6±2.7; p<0.0001) in the Gemcitabine subgroup. Conclusion: PC patients with high hENT1 expression have a better survival outcomes when receiving Gemcitabine. hENT1 expression can be a great prognostic indicator for PC patients to receive Gemcitabine treatment.

Human equilibrative nucleoside transporter 1 (hENT1): do we really have a new predictive biomarker of chemotherapy outcome in pancreatic cancer patients?

Although systemic chemotherapy significantly improves the overall survival of pancreatic cancer patients, the prognosis remains extremely poor. The development of a drug resistance, either de novo or induced resistance, significantly limits the effectiveness of chemotherapy. SLC29A1 gene encodes human equilibrative nucleoside transporter 1 (hENT1) protein that is mediating the transport of nucleotides, both purines and pyrimidines, into the tumor cells. The aim of this mini-review is to summarize the current information concerning the prognostic and predictive role of SLC29A1 transporter (hENT1) expression in pancreatic cancer. Increased expression of SLC29A1 in vitro has been described as a potential critical factor determining the sensitivity of pancreatic cancer cells to gemcitabine and 5-fluorouracil, the principal cytotoxic agents used in the treatment of pancreatic cancer. The reports on the relationship between SLC29A1 expression and prognosis of patients with pancreatic cancer are currently rather conflicting. However, majority of studies on patients with resected pancreatic cancer have suggested that high SLC29A1expression may be predictive of improved survival in patients treated with gemcitabine. SLC29A1 has not been shown to represent a predictive biomarker for patients treated by 5-fluorouracil. In conclusion, potential prognostic and predictive role of SLC29A1 has been demonstrated for selected subset of patients.

Gestational Diabetes-Placental Expression of Human Equilibrative Nucleoside Transporter 1 (hENT1): Is Delayed Villous Maturation an Adaptive Pattern?

Gestational diabetes mellitus (GDM) is a metabolic disease that can affect placental villous maturation and villous vascularity. The main effects of GDM on placental growth are a delay of villous maturation (DVM) and decreased formation of vasculo-syncytial membranes (VSM). Human equilibrative nucleoside transporter-1 (hENT1) is an adenosine transporter expressed in the human umbilical vein endothelial cells (HUVEC) and human placental microvascular endothelium cells (hPMEC). Its role is crucial in maintaining physiological fetal adenosine levels during pregnancy, and its reduction has been described in GDM. Twenty-four placentas from pregnancies with a confirmed diagnosis of GDMd and twenty-four matched non-GDM placentas (controls) were retrospectively analyzed to investigate the immunohistochemical expression of hENT1 in HUVEC and hPMEC. The study included the quantitative evaluation of VSM/mm2 in placental tissue and the immunohistochemical quantitative evaluation of Ki-67, PHH3, and p57 in villous trophoblast. hENT1 expression was higher in all the vascular districts of the control cases compared to the GDMd placentas (p < 0.0001). The VSM/mm2 were lower in the GDMd cases, while the Ki-67, PHH3, and p57 were higher when compared to the control cases. To our knowledge, this is the first report of hENT1 expression in the human placentas of GDM patients. The absence/low expression of hENT1 in all the GDMd patients may indicate a potential role in microvascular adaptative mechanisms. The trophoblasts' proliferative/antiapoptotic pattern (high Ki-67, high PHH3, and high p57 count) may explain the statistically significant lower number of VSM/mm2 found in the GDMd cases.

Clinical use of biomarkers in the field of cytotoxic nucleoside analogues.

OBJECTIVES: Cytotoxic nucleosides (gemcitabine, cytarabine…) are used for the treatment of various malignancies. Their activity is dependent on the interaction with several proteins and enzymes of nucleotide metabolism. It has for a long time been hypothesized that the clinical activity of nucleoside analogues can be predicted by studying corresponding genes or gene products in clinical samples. METHODS: In this short review, I will present old and new published data from our group and others about the prediction of activity of these drugs concentrating on gene-candidate approaches, and discuss biological and technical limitations of these. RESULTS: A large number of studies have been conducted in various clinical settings (drugs, disease, patient cohort…) evaluating DNA, mRNA or protein-related markers. Although some individual parameters and associations thereof have been validated, only a very few numbers have been implemented in pretreatment evaluations of patients. CONCLUSION: There is still much to do in the field of outcome-prediction with nucleoside analogues. The use of multiparametric methods could increase the success rate but at the cost of a poorer understanding of molecular mechanisms.

Prognostic and predictive value of human equilibrative nucleoside transporter 1 (hENT1) in extrahepatic cholangiocarcinoma: a translational study.

Introduction: Effective (neo) adjuvant chemotherapy for cholangiocarcinoma is lacking due to chemoresistance and the absence of predictive biomarkers. Human equilibrative nucleoside transporter 1 (hENT1) has been described as a potential prognostic and predictive biomarker. In this study, the potential of rabbit-derived (SP120) and murine-derived (10D7G2) antibodies to detect hENT1 expression was compared in tissue samples of patients with extrahepatic cholangiocarcinoma (ECC), and the predictive value of hENT1 was investigated in three ECC cell lines. Methods: Tissues of 71 chemonaïve patients with histological confirmation of ECC were selected and stained with SP120 or 10D7G2 to assess the inter-observer variability for both antibodies and the correlation with overall survival. Concomitantly, gemcitabine sensitivity after hENT1 knockdown was assessed in the ECC cell lines EGI-1, TFK-1, and SK-ChA-1 using sulforhodamine B assays. Results: Scoring immunohistochemistry for hENT1 expression with the use of SP120 antibody resulted in the highest interobserver agreement but did not show a prognostic role of hENT1. However, 10D7G2 showed a prognostic role for hENT1, and a potential predictive role for gemcitabine sensitivity in hENT1 in SK-ChA-1 and TFK-1 cells was found. Discussion: These findings prompt further studies for both preclinical validation of the role of hENT1 and histochemical standardization in cholangiocarcinoma patients treated with gemcitabine-based chemotherapy.

Insight into the nucleoside transport and inhibition of human ENT1.

The human equilibrative nucleoside transporter 1 (hENT1) is an effective controller of adenosine signaling by regulating its extracellular and intracellular concentration, and has become a solid drug target of clinical used adenosine reuptake inhibitors (AdoRIs). Currently, the mechanisms of adenosine transport and inhibition for hENT1 remain unclear, which greatly limits the in-depth understanding of its inner workings as well as the development of novel inhibitors. In this work, the dynamic details of hENT1 underlie adenosine transport and the inhibition mechanism of the non-nucleoside AdoRIs dilazep both were investigated by comparative long-time unbiased molecular dynamics simulations. The calculation results show that the conformational transitions of hENT1 from the outward open to metastable occluded state are mainly driven by TM1, TM2, TM7 and TM9. One of the trimethoxyphenyl rings in dilazep serves as the adenosyl moiety of the endogenous adenosine substrate to competitively occupy the orthosteric site of hENT1. Due to extensive and various VDW interactions with N30, M33, M84, P308 and F334, the other trimethoxyphenyl ring is stuck in the opportunistic site near the extracellular side preventing the complete occlusion of thin gate simultaneously. Obviously, dilazep shows significant inhibitory activity by disrupting the local induce-fit action in substrate binding cavity and blocking the transport cycle of whole protein. This study not only reveals the nucleoside transport mechanism by hENT1 at atomic level, but also provides structural guidance for the subsequent design of novel non-nucleoside AdoRIs with enhanced pharmacologic properties.

Looking to the future: biomarkers in the management of pancreatic adenocarcinoma.

The incidence and mortality of pancreas cancer converge. There has been little advancement in the treatment of pancreas cancer since the acceptance of gemcitabine as the standard therapy. Unfortunately, the efficacy of gemcitabine is dismal. While there is much discussion for the development of biomarkers to help direct therapy in this area, there is little action to move them into clinical practice. Herein, we review potential pancreatic cancer biomarkers and discuss the limitations in their implementation.

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.

SF3B1 modulators affect key genes in metastasis and drug influx: a new approach to fight pancreatic cancer chemoresistance.

Aim: Because mutations of splicing factor 3B subunit-1 (SF3B1) have been identified in 4% of pancreatic ductal adenocarcinoma (PDAC) patients, we investigated the activity of new potential inhibitors of SF3B1 in combination with gemcitabine, one of the standard drugs, in PDAC cell lines. Methods: One imidazo[2,1-b][1,3,4]thiadiazole derivative (IS1) and three indole derivatives (IS2, IS3 and IS4), selected by virtual screening from an in-house library, were evaluated by the sulforhodamine-B and wound healing assay for their cytotoxic and antimigratory activity in the PDAC cells SUIT-2, Hs766t and Panc05.04, the latter harbouring the SF3B1 mutations. The effects on the splicing pattern of proto-oncogene recepteur d'origine nantais (RON) and the gemcitabine transporter human equilibrative nucleoside transporter-1 (hENT1) were assessed by PCR, while the ability to reduce tumour volume was tested in spheroids of primary PDAC cells. Results: The potential SF3B1 modulators inhibited PDAC cell proliferation and prompted induction of cell death. All compounds showed an interesting anti-migratory ability, associated with splicing RON/ΔRON shift in SUIT-2 cells after 24 h exposure. Moreover, IS1 and IS4 potentiated the sensitivity to gemcitabine in both conventional 2D monolayer and 3D spheroid cultures, and these results might be explained by the statistically significant increase in hENT1 expression (P < 0.05 vs. untreated control cells), potentially reversing PDAC chemoresistance. Conclusion: These results support further studies on new SF3B1 inhibitors and the role of RON/hENT1 modulation to develop effective drug combinations against PDAC.