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.

Exploring novel dilazep derivatives as hENT1 inhibitors and potentially covalent molecular tools.

The human equilibrative nucleoside transporter 1 (SLC29A1, hENT1) is a solute carrier that modulates the passive transport of nucleosides and nucleobases, such as adenosine. This nucleoside regulates various physiological processes, such as vasodilation and -constriction, neurotransmission and immune defense. Marketed drugs such as dilazep and dipyridamole have proven useful in cardiovascular afflictions, but the application of hENT1 inhibitors can be beneficial in a number of other diseases. In this study, 39 derivatives of dilazep's close analogue ST7092 were designed, synthesized and subsequently assessed using [3H]NBTI displacement assays and molecular docking. Different substitution patterns of the trimethoxy benzoates of ST7092 reduced interactions within the binding pocket, resulting in diminished hENT1 affinity. Conversely, [3H]NBTI displacement by potentially covalent compounds 14b, 14c, and 14d resulted in high affinities (Ki values between 1.1 and 17.5 nM) for the transporter, primarily by the ability of accommodating the inhibitors in various ways in the binding pocket. However, any indication of covalent binding with amino acid residue C439 remained absent, conceivably as a result of decreased nucleophilic residue reactivity. In conclusion, this research introduces novel dilazep derivatives that are active as hENT1 inhibitors, along with the first high affinity dilazep derivatives equipped with an electrophilic warhead. These findings will aid the rational and structure-based development of novel hENT1 inhibitors and pharmacological tools to study hENT1's function, binding mechanisms, and its relevance in (patho)physiological conditions.

Tailored adjuvant gemcitabine versus 5-fluorouracil/folinic acid based on hENT1 immunohistochemical staining in resected pancreatic ductal adenocarcinoma: A biomarker stratified prospective trial.

BACKGROUND: The study aimed to evaluate the clinical outcomes of tailored adjuvant chemotherapy according to human equilibrative nucleoside transporter 1 (hENT1) expression in resected pancreatic ductal adenocarcinoma (PDA). METHODS: Patients who underwent pancreatectomy for PDA were enrolled prospectively. According to intra-tumoral hENT1 expression, the high hENT1 (≥50%) group received gemcitabine and the low hENT1 (<50%) group received 5-fluorouracil plus folinic acid (5-FU/FA). The propensity score-matched control consisted of patients who received hENT1-independent adjuvant chemotherapy. The primary outcome was recurrence free survival (RFS) and the secondary outcomes were overall survival (OS) and toxicities. RESULTS: Between May 2015 and June 2017, we enrolled 44 patients with resected PDA. During a median follow-up period of 28.5 months, the intention-to-treat population showed much longer median RFS [22.9 (95% CI, 11.3-34.5) vs. 10.9 (95% CI, 6.9-14.9) months, P = 0.043] and median OS [36.2 (95% CI, 26.5-45.9) vs. 22.1 (95% CI, 17.7-26.6) months, P = 0.001] compared to the controls. Among 5 patients in the low hENT1 group who discontinued treatment, 2 patients receiving 5-FU/FA discontinued treatment due to drug toxicities (febrile neutropenia and toxic epidermal necrolysis). CONCLUSION: Tailored adjuvant chemotherapy based on hENT1 staining provides excellent clinical outcomes among patients with resected PDA. CLINICAL TRIAL REGISTRATION: clinicaltrials.gov identifier: NCT02486497.

Staphylococcus aureus targets the purine salvage pathway to kill phagocytes.

Staphylococcus aureus colonizes large segments of the human population and causes invasive infections due to its ability to escape phagocytic clearance. During infection, staphylococcal nuclease and adenosine synthase A convert neutrophil extracellular traps to deoxyadenosine (dAdo), which kills phagocytes. The mechanism whereby staphylococcal dAdo intoxicates phagocytes is not known. Here we used CRISPR-Cas9 mutagenesis to show that phagocyte intoxication involves uptake of dAdo via the human equilibrative nucleoside transporter 1, dAdo conversion to dAMP by deoxycytidine kinase and adenosine kinase, and signaling via subsequent dATP formation to activate caspase-3-induced cell death. Disruption of this signaling cascade confers resistance to dAdo-induced intoxication of phagocytes and may provide therapeutic opportunities for the treatment of infections caused by antibiotic-resistant S. aureus strains.

Human equilibrative nucleoside transporter-1 expression is a predictor in patients with resected pancreatic cancer treated with adjuvant S-1 chemotherapy.

The high expression of human equilibrative nucleoside transporter-1 (hENT1) and the low expression of dihydropyrimidine dehydrogenase (DPD) are reported to predict a favorable prognosis in patients treated with gemcitabine (GEM) and 5-fluorouracil (5FU) as the adjuvant setting, respectively. The expression of hENT1 and DPD were analyzed in patients registered in the JASPAC 01 trial, which showed a better survival of S-1 over GEM as adjuvant chemotherapy after resection for pancreatic cancer, and their possible roles for predicting treatment outcomes and selecting a chemotherapeutic agent were investigated. Intensity of hENT1 and DPD expression was categorized into no, weak, moderate or strong by immunohistochemistry staining, and the patients were classified into high (strong/moderate) and low (no/weak) groups. Specimens were available for 326 of 377 (86.5%) patients. High expression of hENT1 and DPD was detected in 100 (30.7%) and 63 (19.3%) of 326 patients, respectively. In the S-1 arm, the median overall survival (OS) with low hENT1, 58.0 months, was significantly better than that with high hENT1, 30.9 months (hazard ratio 1.75, P = 0.007). In contrast, there were no significant differences in OS between DPD low and high groups in the S-1 arm and neither the expression levels of hENT1 nor DPD revealed a relationship with treatment outcomes in the GEM arm. The present study did not show that the DPD and hENT1 are useful biomarkers for choosing S-1 or GEM as adjuvant chemotherapy. However, hENT1 expression is a significant prognostic factor for survival in the S-1 arm.

Human equilibrative nucleoside transporter-1 and deoxycytidine kinase can predict gemcitabine effectiveness in Egyptian patients with Hepatocellular carcinoma.

BACKGROUND: Several biomarkers of gemcitabine effectiveness have been studied in cancers, but less so in hepatocellular carcinoma (HCC), which is identified as the fifth most common cancer worldwide. Investigation of human equilibrative nucleoside transporter-1 (HENT-1) and deoxycytidine kinase (DCK), genes involved in gemcitabine uptake and metabolism, can be beneficial in the selection of potential cancer patients who could be responding to the treatment. AIM: To study HENT-1 and DCK gene expression in HCC patients with different protocols of treatment. METHODS: Using real-time PCR, we analyzed expression levels of HENT-1 and DCK genes from peripheral blood samples of 109 patients (20 controls & 89 HCC patients) between March 2015 and March 2017. All the 89 HCC patients received the antioxidants selenium (Se) and vitamin E (Vit.E) either alone (45 patients) or in combination with gemcitabine (24 patients) or radiofrequency ablation (RFA) (20 patients). RESULTS: There was a significant increase in HENT-1 expression levels in HCC patients treated with Se and Vit.E alone as compared to controls (P Ë‚ .0001), while there was no significant difference between HCC patients treated with gemcitabine or RFA as compared to controls. In contrast, expression of DCK was significantly increased in all groups of HCC patients as compared to controls (P Ë‚ .0001). CONCLUSIONS: HENT-1 and DCK mRNA expressions are important markers of HCC and for GEM effect and GEM sensitivity in patients with HCC. This could be beneficial in the selection of HCC patients sensitive to gemcitabine to avoid subjecting resistant patients to unnecessary chemotherapy.

Role of hepatocyte nuclear factor 4 alpha in cell proliferation and gemcitabine resistance in pancreatic adenocarcinoma.

BACKGROUND: Hepatocyte nuclear factor 4α (HNF4α) is a tissue-specific transcription factor that regulates the expression of numerous genes in hepatocytes and pancreatic ß cells. HNF4α has been reported to affect cell proliferation and chemoresistance in several cancers. However, the role of HNF4α in pancreatic adenocarcinoma (PDAC) has not been studied extensively and remains unclear. METHODS: By utilizing immunohistochemical (IHC) staining, we measured the expression of HNF4α in PDAC tissues. By silencing HNF4α in PDAC cell lines, we assessed the impact of HNF4α on pancreatic cancer cell proliferation and gemcitabine sensitivity. We used CCK8 and colony formation assays to examine the effect of HNF4α on cell proliferation. A flow cytometry assay was used to assess cell apoptosis. The expression of gemcitabine-related genes was detected by quantitative real­time PCR (qRT-PCR) and Western blotting. IHC was utilized to assess the correlation between HNF4α and human equilibrative nucleoside transporter 1 (hENT1) expression in PDAC patients. Chromatin immunoprecipitation (ChIP) and dual­luciferase reporter assays were used to confirm that hENT1 is a target gene of HNF4α. RESULTS: Increased HNF4α expression was detected in PDAC tissues; patients with higher HNF4α expression displayed worse prognosis. To elucidate the function of HNF4α, we examined its role in pancreatic cancer cell proliferation, apoptosis and gemcitabine resistance. In HNF4α-silenced Capan-1 and MiaPaCa-2 cells, we observed decreased cell proliferation and increased sensitivity to gemcitabine compared to those of controls. The mechanism of HNF4α in gemcitabine-related chemosensitivity was then explored. In response to HNF4α silencing, the expression levels of gemcitabine-related proteins, hENT1 and deoxycytidine kinase (dCK) were significantly increased. Additionally, hENT1 was negatively correlated with HNF4α in PDAC tissue samples. Moreover, we identified hENT1 as a downstream target of HNF4α. CONCLUSION: HNF4α is a prognostic marker for overall survival, is required for pancreatic cancer cell proliferation and promotes resistance to gemcitabine by downregulating hENT1. Therefore, targeting HNF4α might reverse gemcitabine resistance and provide novel treatment strategies for PDAC.

GnT-V promotes chemosensitivity to gemcitabine in bladder cancer cells through ß1,6 GlcNAc branch modification of human equilibrative nucleoside transporter 1.

Human equilibrative nucleoside transporter 1 (hENT1) transports nucleoside analogue drugs across cellular membranes and is necessary for the uptake of many anti-tumor drugs. Gemcitabine is a frontline agent of chemotherapy for bladder cancer despite its limited efficacy due to chemoresistance, there is an acute need to decipher mechanisms underlying chemosensitivity to gemcitabinein in bladder cancer cells. Here we report a novel role for N-acetylglucosaminyltransferase V (GnT-V) in gemcitabine chemosensitivity. In this study, we found that GnT-V expression affected cell death rate to gemcitabine in different bladder cancer cells and down-regulation of GnT-V inhibited the gemcitabine sensitivity with time and dose dependent way in T24 cells. Moreover, mechanistic investigations showed that silencing GnT-V caused dramatic decrease of ß1,6 GlcNAc structure on hENT1 leading to apparently decreased accumulation of hENT1 at plasma membrane, and therefore result in less uptake of gemcitabine in T24/shRNA cells. Together, our present study indicated that GnT-V enhances gemcitabine chemosensitivity via modulation of hENT1 N-glycosylation and transport activity in T24 cells, providing new insights into how N-glycosylation drives antitumor drug sensitivity during chemotherapy for patients with cancer.

Current and future biomarkers for pancreatic adenocarcinoma.

Although pancreatic cancer is only the twelfth most common type of cancer in the world, it features a very unfavorable prognosis. The mortality rate almost equals the incidence rate, corroborating the very poor prognosis of pancreatic cancer. The 5-year survival rate for all stages of pancreatic ductal adenocarcinoma is only 7%. Surgical resection represents the only potentially curative treatment option for pancreatic ductal adenocarcinoma patients but is often not feasible due to the advanced stage of the disease upon diagnosis. For advanced disease, palliative chemotherapy is the treatment of choice although the regimens available to date are untargeted and have extensive side-effect profiles, making them unsuitable for patients with a low performance status. For this reason, early detection of pancreatic cancer is essential in order to provide patients with an optimal therapeutic approach. Up to the present day, carbohydrate antigen 19-9 is the only diagnostic marker approved by the U.S. Food and Drug Administration but its diagnostic potential is limited due to its restricted sensitivity and specificity, supporting the urgent need for novel biomarkers. In addition, prognostic and treatment-predictive biomarkers might provide essential information regarding personalized treatment decisions for individual patients. In this article, we aim to review current and future diagnostic, prognostic, and treatment-predictive biomarkers for pancreatic cancer.

Membrane Localization of Human Equilibrative Nucleoside Transporter 1 in Tumor Cells May Predict Response to Adjuvant Gemcitabine in Resected Cholangiocarcinoma Patients.

BACKGROUND: The use of gemcitabine as an adjuvant modality for cholangiocarcinoma (CC) is increasing, but limited data are available on predictive biomarkers of response. Human equilibrative nucleoside transporter 1 (hENT-1) is the major transporter involved in gemcitabine intracellular uptake. This study investigated the putative predictive role of hENT-1 localization in tumor cells of CC patients undergoing treatment with adjuvant gemcitabine. METHODS: Seventy-one consecutive patients with resected CC receiving adjuvant gemcitabine at our center were retrospectively analyzed by immunohistochemistry for hENT-1 localization in tumor cells. The main outcome measure was disease-free survival (DFS). Hazard ratios (HRs) of relapse and associated 95% confidence intervals (CIs) were obtained from proportional hazards regression models stratified on quintiles of propensity score. RESULTS: Twenty-three (32.4%) cases were negative for hENT-1, 22 (31.0%) were positive in the cytoplasm only, and 26 (36.6%) showed concomitant cytoplasm/membrane staining. Patients with membrane hENT-1 had a longer DFS (HR 0.49, 95% CI 0.24-0.99, p = .046) than those who were negative or positive only in the cytoplasm of tumor cells. Notably, the association between DFS and membrane hENT-1 was dependent on the number of gemcitabine cycles (one to two cycles: HR 0.96, 95% CI 0.34-2.68; three to four cycles: HR 0.99, 95% CI 0.34-2.90; five to six cycles: HR 0.27, 95% CI 0.10-0.77). CONCLUSION: hENT-1 localization on tumor cell membrane may predict response to adjuvant gemcitabine in CC patients receiving more than four cycles of chemotherapy. Further prospective randomized trials on larger populations are required to confirm these preliminary results, so that optimal gemcitabine-based chemotherapy may be tailored for CC patients in the adjuvant setting. IMPLICATIONS FOR PRACTICE: Gemcitabine is becoming an increasingly used adjuvant modality in cholangiocarcinoma (CC), but limited data are available on predictive biomarkers of response. In this study, patients receiving more than four cycles of adjuvant gemcitabine and harboring Human equilibrative nucleoside transporter 1 (hENT-1, the major transporter involved in gemcitabine intracellular uptake) on tumor cell membrane had a longer disease-free survival compared with patients negative or positive for hENT-1 only in the cytoplasm of tumor cells. Overall these results may lay the basis for further prospective randomized trials based on a larger population of patients and may prove useful for tailoring appropriate gemcitabine-based chemotherapy for CC patients in the adjuvant setting.

Immunohistochemical expression profiles of solute carrier transporters in alpha-fetoprotein-producing gastric cancer.

AIMS: Alpha-fetoprotein (AFP)-producing gastric cancer (GC) is an aggressive tumour with high rates of liver metastasis and poor prognosis, and for which a validated chemotherapy regimen has not been established. Drug uptake by solute carrier (SLC) transporters is proposed as one of the mechanisms involved in sensitivity to chemotherapy. In this study, we aimed to develop important insights into effective chemotherapeutic regimens for AFP-producing GC. METHODS AND RESULTS: We evaluated immunohistochemically the expression levels of a panel of SLC transporters in 20 AFP-producing GCs and 130 conventional GCs. SLC transporters examined were human equilibrative nucleoside transporter 1 (hENT1), organic anion transporter 2 (OAT2), organic cation transporter (OCT) 2, OCT6 and organic anion-transporting polypeptide 1B3 (OATP1B3). The rates of high expression levels of hENT1 (hENT1high ) and OAT2 (OAT2high ) were statistically higher in AFP-producing GC, compared with conventional GC. When analysing hENT1 and OAT2 in combination, hENT1high /OAT2high was the most particular expression profile for AFP-producing GC, with a greater significance than hENT1 or OAT2 alone. However, no significant differences in OCT2, OCT6 or OATP1B3 levels were detected between AFP-producing and conventional GCs. However, immunoreactivity for hENT1, OAT2 and OCT6 tended to be increased in GC tissues compared with non-neoplastic epithelia. CONCLUSIONS: Because hENT1 and OAT2 are crucial for the uptake of gemcitabine and 5-fluorouracil, respectively, our results suggest that patients with AFP-producing GC could potentially benefit from gemcitabine/fluoropyrimidine combination chemotherapy. Increased expression of hENT1, OAT2 and OCT6 may also be associated with the progression of GC.

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.

Concordance of human equilibrative nucleoside transporter-1 expressions between murine (10D7G2) and rabbit (SP120) antibodies and association with clinical outcomes of adjuvant chemotherapy for pancreatic cancer: A collaborative study from the JASPAC 01 trial.

BACKGROUND: Expression of human equilibrative nucleoside transporter-1 (hENT1) is reported to predict survival of gemcitabine (GEM)-treated patients. However, predictive values of immunohistochemical hENT1 expression may differ according to the antibodies, 10D7G2 and SP120. AIM: We aimed to investigate the concordance of immunohistochemical hENT1 expression between the two antibodies and prognosis. METHODS: The subjects of this study were totally 332 whose formalin-fixed paraffin-embedded specimens and/or unstained sections were obtained. The individual H-scores and four classifications according to the staining intensity were applied for the evaluation of hENT1 expression by 10D7G2 and SP120, respectively. RESULTS: The highest concordance rate (79.8%) was obtained when the cut-off between high and low hENT1 expression using SP120 was set between moderate and strong. There were no correlations of hENT1 mRNA level with H-score (p = .258). Although the hENT1 mRNA level was significantly different among four classifications using SP120 (p = .011), there was no linear relationship among them. Multivariate analyses showed that adjuvant GEM was a significant predictor of the patients with low hENT1 expression using either 10D7G2 (Hazard ratio [HR] 2.39, p = .001) or SP120 (HR 1.84, p < .001). In contrast, agent for adjuvant chemotherapy was not significant predictor for the patients with high hENT1 expression regardless of the kind of antibody. CONCLUSION: The present study suggests that the two antibodies for evaluating hENT1 expression are equivalent depending on the cut-off point and suggests that S-1 is the first choice of adjuvant chemotherapy for pancreatic cancer with low hENT1 expression, whereas either S-1 or GEM can be introduced for the pancreatic cancer with high hENT1 expression, no matter which antibody is used.

"Open Sesame?": Biomarker Status of the Human Equilibrative Nucleoside Transporter-1 and Molecular Mechanisms Influencing its Expression and Activity in the Uptake and Cytotoxicity of Gemcitabine in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive tumor characterized by early invasiveness, rapid progression and resistance to treatment. For more than twenty years, gemcitabine has been the main therapy for PDAC both in the palliative and adjuvant setting. After the introduction of FOLFIRINOX as an upfront treatment for metastatic disease, gemcitabine is still commonly used in combination with nab-paclitaxel as an alternative first-line regimen, as well as a monotherapy in elderly patients unfit for combination chemotherapy. As a hydrophilic nucleoside analogue, gemcitabine requires nucleoside transporters to permeate the plasma membrane, and a major role in the uptake of this drug is played by human equilibrative nucleoside transporter 1 (hENT-1). Several studies have proposed hENT-1 as a biomarker for gemcitabine efficacy in PDAC. A recent comprehensive multimodal analysis of hENT-1 status evaluated its predictive role by both immunohistochemistry (with five different antibodies), and quantitative-PCR, supporting the use of the 10D7G2 antibody. High hENT-1 levels observed with this antibody were associated with prolonged disease-free status and overall-survival in patients receiving gemcitabine adjuvant chemotherapy. This commentary aims to critically discuss this analysis and lists molecular factors influencing hENT-1 expression. Improved knowledge on these factors should help the identification of subgroups of patients who may benefit from specific therapies and overcome the limitations of traditional biomarker studies.

Attempts to remodel the pathways of gemcitabine metabolism: Recent approaches to overcoming tumours with acquired chemoresistance.

Gemcitabine is a cytidine analogue frequently used in the treatment of various cancers. However, the development of chemoresistance limits its effectiveness. Gemcitabine resistance is regulated by various factors, including aberrant genetic and epigenetic controls, metabolism of gemcitabine, the microenvironment, epithelial-to-mesenchymal transition, and acquisition of cancer stem cell properties. In many situations, results using cell lines offer valuable lessons leading to the first steps of important findings. In this review, we mainly discuss the factors involved in gemcitabine metabolism in association with chemoresistance, including nucleoside transporters, deoxycytidine kinase, cytidine deaminase, and ATP-binding cassette transporters, and outline new perspectives for enhancing the efficacy of gemcitabine to overcome acquired chemoresistance.

New Imidazo[2,1-b][1,3,4]Thiadiazole Derivatives Inhibit FAK Phosphorylation and Potentiate the Antiproliferative Effects of Gemcitabine Through Modulation of the Human Equilibrative Nucleoside Transporter-1 in Peritoneal Mesothelioma.

BACKGROUND/AIM: A new class of imidazo[2,1-b][1,3,4]thiadiazole compounds have recently been evaluated as inhibitors of phosphorylation of focal adhesion kinase (FAK) in pancreatic cancer. FAK is overexpressed in mesothelioma and has recently emerged as an interesting target for the treatment of this disease. MATERIALS AND METHODS: Ten imidazo[2,1-b][1,3,4]thiadiazole compounds characterized by indole bicycle and a thiophene ring, were evaluated for their cytotoxic activity in two primary cell cultures of peritoneal mesothelioma, MesoII and STO cells. RESULTS: Compounds 1a and 1b showed promising antitumor activity with IC50 values in the range of 0.59 to 2.81 µM in both cell lines growing as monolayers or as spheroids. Their antiproliferative and antimigratory activity was associated with inhibition of phospho-FAK, as detected by a specific ELISA assay in STO cells. Interestingly, these compounds potentiated the antiproliferative activity of gemcitabine, and these results might be explained by the increase in the mRNA expression of the key gemcitabine transporter human equilibrative nucleoside transporter-1 (hENT-1). CONCLUSION: These promising results support further studies on new imidazo[2,1-b][1,3,4]thiadiazole compounds as well as on the role of both FAK and hENT-1 modulation in order to develop new drug combinations for peritoneal mesothelioma.

Development of gemcitabine-resistant patient-derived xenograft models of pancreatic ductal adenocarcinoma.

AIM: Gemcitabine is a frontline agent for locally-advanced and metastatic pancreatic ductal adenocarcinoma (PDAC), but neither gemcitabine alone nor in combination produces durable remissions of this tumor type. We developed three PDAC patient-derived xenograft (PDX) models with gemcitabine resistance (gemR) acquired in vivo, with which to identify mechanisms of resistance relevant to drug exposure in vivo and to evaluate novel therapies. METHODS: Mice bearing independently-derived PDXs received 100 mg/kg gemcitabine once or twice weekly. Tumors initially responded, but regrew on treatment and were designated gemR. We used immunohistochemistry to compare expression of proteins previously associated with gemcitabine resistance [ribonucleotide reductase subunit M1 (RRM1), RRM2, human concentrative nucleoside transporter 1 (hCNT1), human equilibrative nucleoside transporter 1 (hENT1), cytidine deaminase (CDA), and deoxycytidine kinase (dCK)] in gemR and respective gemcitabine-naive parental tumors. RESULTS: Parental and gemR tumors did not differ in tumor cell morphology, amount of tumor-associated stroma, or expression of stem cell markers. No consistent pattern of expression of the six gemR marker proteins was observed among the models. Increases in RRM1 and CDA were consistent with in vitro-derived gemR models. However, rather than the expected decreases of hCNT1, hENT1, and dCK, gemR tumors expressed no change in or higher levels of these gemR marker proteins than parental tumors. CONCLUSION: These models are the first PDAC PDX models with gemcitabine resistance acquired in vivo. The data indicate that mechanisms identified in models with resistance acquired in vitro are unlikely to be the predominant mechanisms when resistance is acquired in vivo. Ongoing work focuses on characterizing unidentified mechanisms of gemR and on identifying agents with anti-tumor efficacy in these gemR models.

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.

Membrane human equilibrative nucleoside transporter 1 is associated with a high proliferation rate and worse survival in resected intrahepatic cholangiocarcinoma patients not receiving adjuvant treatments.

Human equilibrative nucleoside transporter 1 (hENT-1) is a membrane nucleoside transporter mediating the intracellular uptake of nucleosides and their analogues. hENT-1 was recently reported to have a predictive role in intrahepatic cholangiocarcinoma (iCC) patients receiving adjuvant gemcitabine-based chemotherapy, but its biological and clinical significance in iCC remains unsettled. This study investigated the role of hENT-1 in regulating tumour growth and predicting the survival of 40 resected iCC patients not receiving adjuvant treatments. hENT-1 expression was found to be significantly higher in iCC than in the matched non-tumoural liver. Patients harbouring hENT-1 localised on the tumour cell membrane had a worse overall survival than membrane hENT-1-negative patients (median 21.2 months vs 30.3 months, p = 0.031), with an adjusted hazard ratio of 2.8 (95% confidence interval 1.01-7.76). Moreover, membrane hENT-1-positive patients had a higher percentage of Ki67-positive cells in tumour tissue than membrane hENT-1-negative patients (median 23% vs 5%, p < 0.0001). Functional analyses in iCC cell lines revealed that hENT-1 silencing inhibited cell proliferation and induced apoptosis in HUH-28 cells expressing hENT-1 on the cell membrane, but not in SNU-1079 cells expressing the transporter only in the cytoplasm. Overall, these findings suggest that membrane hENT-1 is involved in iCC proliferation and associated with worse survival in resected iCC patients. Further prospective studies on larger cohorts are required to confirm these results and better define the potential prognostic role of membrane hENT-1 in this setting of patients.

Prognostic immunohistochemical biomarkers of chemotherapy efficacy in biliary tract cancer: A systematic review and meta-analysis.

INTRODUCTION: Chemotherapy is the mainstay of systemic treatment of biliary tract cancer (BTC). However, the treatment response to chemotherapy varies between patients. Currently, no prognostic biomarkers for chemotherapy efficacy have been considered for use in clinical practice. A systematic review was conducted to evaluate the prognostic value of immunohistochemical biomarkers for chemotherapy in patients with resected as well as with advanced BTC. METHOD: Medline and EMBASE databases were searched up to March 2017 for studies that evaluated biomarker expression by immunohistochemistry in resected or advanced BTC patients treated with chemotherapy. The primary endpoints were overall survival (OS) and disease or progression free survival (DFS or PFS). RESULT: Twenty-six studies, including a total of 1348 patients and 26 different biomarkers, met the inclusion criteria and were included in this review. The most frequently studied prognostic biomarkers in BTC were the human Equilibrative Nucleoside Transporter 1 (hENT1), Ribonucleotide Reductase M1 (RRM1), and excision repair cross-complementation 1 (ERCC1). In the meta-analysis of patients treated with gemcitabine-based chemotherapy, high hENT1 expression was associated with longer OS (HR 0.43, 95% CI: 0.28 to 0.64) and DFS/PFS (HR 0.45, 95% CI: 0.33 to 0.61). CONCLUSION: hENT1 is a promising prognostic biomarker for gemcitabine-based chemotherapy in resected as well as in advanced BTC and should be further validated for the selection of patients for chemotherapy.