[Corrigendum] Dexamethasone-induced inhibition of miR-132 via methylation promotes TGF-ß-driven progression of pancreatic cancer.

Subsequently to the publication of the above article, an interested reader drew to the authors' attention that two pairs of the culture plate images in Fig. 4A-C on p. 60 appeared to be the same, although the images were shown in different orientations; moreover, the 'NC/0 and DEX+miR132' and 'DEX and miR132' pairings of images in the scratch-wound assay experiments shown in Fig. 4B also appeared to be overlapping, such that these were apparently derived from the same original source where the results of differently performed experiments were intended to have been portrayed. After re­examining their original data, the authors have realized that some of the data in Fig. 4A and B were inadvertently assembled incorrectly. The revised version of Fig. 4, showing all the correct data for the culture plate images in Fig. 4A-C (specifically, the images fifth along on the right for Fig. 4B and C have been revised) and the correct images for 'NC/0' and 'DEX/0' in Fig. 4D is shown on on the next page. The authors are grateful to the Editor of International Journal of Oncology for allowing them this opportunity to publish a Corrigendum, and all the authors agree with its publication. Furthermore, the authors apologize to the readership for any inconvenience caused. [International Journal of Oncology 54: 53­64, 2019; DOI: 10.3892/ijo.2018.4616].

Glucocorticoid-induced microRNA-378 signaling mediates the progression of pancreatic cancer by enhancing autophagy.

Glucocorticoids (GCs) are widely used in tumor therapy to reduce tumor growth, inflammation, edema, and other side effects. Controversially, GCs may also cause the progression of highly aggressive pancreatic ductal adenocarcinoma (PDAC). Because microRNA (miR) and autophagy signaling support the invasive growth of PDAC, we asked whether these mechanisms may be targeted by GCs. Six established human PDAC cell lines, tissue from patients who received GC medication (n = 35) prior to surgery, or not (n = 35), and tumor xenografts were examined by RT‒qPCR, transmission electron microscopy (TEM), monodansylcadaverine (MDC) staining, immunohistochemistry, in situ hybridization, gene array and Kaplan‒Meier analysis with bioinformatics, and MTT, western blot, colony, spheroid, migration, and invasion assays. We found that various GCs, including dexamethasone (DEX), induced typical features of macroautophagy with the appearance of autolysosomes, enhanced LC3-II, decreased SQSTM1/p62 expression and induced epithelial-mesenchymal transition (EMT) and gemcitabine resistance. The GC receptor (GR) antagonist mifepristone (RU486) counteracted DEX-induced autophagy features, suggesting that the GC-GR complex is involved in the induction of autophagy. The autophagy-related miR-378i and miR-378a-3p were selected as the top upregulated candidates, and their high expression in PDAC patient tissue correlated with low survival. siRNA-mediated downregulation of miR-378 inhibited DEX-induced autophagy, and tumor progression. Bioinformatics confirmed the contribution of miR-378 to the regulation of signaling networks involved in GC-induced autophagy and tumor progression. The construction of a molecular docking model revealed stable binding of miR-378 to the DEX-GR complex, suggesting direct regulation. These substantial, novel, in-depth data reveal that GCs favor autophagy-mediated cancer progression by inducing miR-378 and GR binding and implicate GR and miR-378 as new therapeutic targets.

Tumor and stroma COL8A1 secretion induces autocrine and paracrine progression signaling in pancreatic ductal adenocarcinoma.

Several collagen subtypes are involved in pancreatic ductal adenocarcinoma (PDAC) desmoplasia, which constrains therapeutic efficacy. We evaluated collagen type VIII alpha 1 chain (COL8A1), whose function in PDAC is currently unknown. We identified COL8A1 expression in 7 examined PDAC cell lines by microarray analysis, western blotting, and RT‒qPCR. Higher COL8A1 expression occurred in 2 gemcitabine-resistant PDAC cell lines; pancreas tissue (n=15) from LSL-KrasG12D/+; p48-Cre mice with advanced PDAC predisposition; and PDAC parenchyma and stroma of a patient tissue microarray (n=82). Bioinformatic analysis confirmed higher COL8A1 expression in PDAC patient tissue available from TCGA (n=183), GTEx (n=167), and GEO (n=261) databases. siRNA or lentiviral sh-mediated COL8A1 inhibition in PDAC cells reduced migration, invasion and gemcitabine resistance and resulted in lower cytidine deaminase and thymidine kinase 2 expression and was rescued by COL8A1-secreting cancer-associated fibroblasts (CAFs). The activation of COL8A1 expression involved cJun/AP-1, as demonstrated by CHIP assay and siRNA inhibition. Downstream of COL8A1, activation of ITGB1 and DDR1 receptors and PI3K/AKT and NF-κB signaling occurred, as detected by expression, adhesion and EMSA binding studies. Orthotopic transplantation of PDAC cells with downregulated COL8A1 expression resulted in reduced tumor xenograft growth and lower gemcitabine resistance but was prevented by cotransplantation of COL8A1-secreting CAFs. Most importantly, COL8A1 expression in PDAC patient tissues from our clinic (n=84) correlated with clinicopathological data, and we confirmed these findings by the use of patient data (n=177) from the TCGA database. These findings highlight COL8A1 expression in tumor and stromal cells as a new biomarker for PDAC progression.

Nanosilver inhibits the progression of pancreatic cancer by inducing a paraptosis-like mixed type of cell death.

Silver has been in clinical use since ancient times and silver nanoparticles (AgNPs) have attracted attention in cancer therapy. We investigated the mechanisms by which AgNPs inhibit pancreatic ductal adenocarcinoma (PDAC). AgNPs were synthesized and 3 human PDAC and 2 nonmalignant primary cell lines were treated with AgNPs. MTT, MAPK, colony, spheroid and scratch assays, Western blotting, TEM, annexin V, 7-AAD, and H2DCFDA staining, FACS analysis, mRNA array and bioinformatics analyses, tumor xenograft transplantation, and immunohistochemistry of the treated cells were performed. We found that minimal AgNPs amounts selectively eradicated PDAC cells within a few hours. AgNPs inhibited cell migration and spheroid and colony formation, damaged mitochondria, and induced paraptosis-like cell death with the presence of cytoplasmic vacuoles, dilation of the ER and mitochondria, ROS formation, MAPK activity, and p62 and LC3b expression, whereas effects on the nucleus, DNA fragmentation, or caspases were not detectable. AgNPs strongly decreased tumor xenograft growth without side effects and reduced the expression of markers for proliferation and DNA repair, but upregulated paraptosis markers. The results highlight nanosilver as complementary agent to improve the therapeutic efficacy in pancreatic cancer.

UHMK1 Is a Novel Marker for Personalized Prediction of Pancreatic Cancer Prognosis.

Pancreatic ductal adenocarcinoma (PDAC) is among the leading causes of cancer mortality, and new therapeutic options are urgently needed. Long noncoding RNA H19 (H19) is known to promote PDAC progression, but the downstream genes of H19 are largely unknown. Five PDAC cell lines, nonmalignant pancreatic cells, TCGA, GEO-derived pancreatic tissues (malignant, n=413; nonmalignant, n=234), a pancreatic tissue array (n=96), and pancreatic tissues from our clinic (malignant, n=20; nonmalignant, n=20) were examined by a gene array, RT-qPCR, Western blotting, MTT, colony formation, wound-healing, siRNA-mediated gene silencing, bioinformatics, xenotransplantation, and immunohistochemistry assays. The cell cycle inhibitor, UHMK1, was identified to have the strongest correlation with H19. UHMK1 expression was enhanced in PDAC, and high UHMK1 expression correlated with tumor stage, and lower overall survival. siRNA-mediated UHMK1 downregulation inhibited progression signaling. siRNA-mediated downregulation of H19 or UHMK1 inhibited tumor proliferation and xenograft growth. Based on the correlation between UHMK1 expression and clinical parameters, we developed a nomogram that reliably predicts patient prognosis and overall survival. Together, we characterized UHMK1 as an H19-induced oncogene and verified it as a novel PDAC prognostic marker for overall survival.

Sulforaphane Targets TRA-1/GLI Upstream of DAF-16/FOXO to Promote C. elegans Longevity and Healthspan.

Broccoli-derived isothiocyanate sulforaphane inhibits inflammation and cancer. Sulforaphane may support healthy aging, but the underlying detailed mechanisms are unclear. We used the C. elegans nematode model to address this question. Wild-type and 4 mutant C. elegans worm strains were fed in the presence or absence of sulforaphane and E. coli food bacteria transfected with RNA interference gene constructs. Kaplan-Meier survival analysis, live imaging of mobility and pharyngeal pumping, fluorescence microscopy, RT-qPCR, and Western blotting were performed. In the wild type, sulforaphane prolonged lifespan and increased mobility and food intake because of sulforaphane-induced upregulation of the sex-determination transcription factor TRA-1, which is the ortholog of the human GLI mediator of sonic hedgehog signaling. In turn, the tra-1 target gene daf-16, which is the ortholog of human FOXO and the major mediator of insulin/IGF-1 and aging signaling, was induced. By contrast, sulforaphane did not prolong lifespan and healthspan when tra-1 or daf-16 was inhibited by RNA interference or when worms with a loss-of-function mutation of the tra-1 or daf-16 genes were used. Conversely, the average lifespan of C. elegans with hyperactive TRA-1 increased by 8.9%, but this longer survival was abolished by RNAi-mediated inhibition of daf-16. Our data suggest the involvement of sulforaphane in regulating healthy aging and prolonging lifespan by inducing the expression and nuclear translocation of TRA-1/GLI and its downstream target DAF-16/FOXO.

Alpha-Lipoic Acid Prevents Side Effects of Therapeutic Nanosilver without Compromising Cytotoxicity in Experimental Pancreatic Cancer.

Silver nanoparticles (AgNPs) have attracted attention in cancer therapy and might support the treatment of pancreatic ductal adenocarcinoma (PDAC). Silver is in clinical use in wound dressings, catheters, stents and implants. However, the side effects of systemic AgNP treatment due to silver accumulation limit its therapeutic application. We evaluated whether the antioxidant and natural agent α-lipoic acid might prevent these side effects. We synthesized AgNPs using an Ionic-Pulser® Pro silver generator and determined the concentration by inductively coupled plasma-optical emission spectrometry. The effect of α-lipoic acid was examined in four PDAC and two nonmalignant cell lines by MTT, FACS analysis, TEM, xenotransplantation and immunohistochemistry. The viability of PDAC cells was nearly totally abolished by AgNP treatment, whereas nonmalignant cells largely resisted. α-Lipoic acid prevented AgNP-induced cytotoxicity in nonmalignant cells but not in PDAC cells, which might be due to the higher sensitivity of malignant cells to silver-induced cytotoxicity. α-Lipoic acid protected mitochondria from AgNP-induced damage and led to precipitation of AgNPs. AgNPs reduced the growth of tumor xenografts, and cotreatment with α-lipoic acid protected chick embryos from AgNP-induced liver damage. Together, α-lipoic acid strongly reduced AgNP-induced side effects without weakening the therapeutic efficacy.

Therapy of pancreatic cancer with alternating electric fields: Limitations of the method.

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant tumor with a poor prognosis. More effective treatment options are urgently needed. The use of physical and weak alternating electric fields (TTFields) can inhibit cell division of PDAC carcinoma and is currently being investigated in clinical trials. Here, we analyzed this new physical treatment under non-ideal conditions such as may occur during patient treatment. Three established human PDAC cell lines BxPC-3, gemcitabine-resistant BxPC-3 (BxGem), AsPC-1, and a non-malignant primary pancreatic cell line CRL-4023 were treated with TTFields in vitro. MTT assays, electrical impedance measurement, cell staining with Annexin V/7AAD followed by FACS analysis, digital image analysis and immunohistochemistry were performed. Treatment with TTFields smaller than 0.7 V/cm and field lines in the direction of mitotic spindle orientation significantly inhibited proliferation of all PDAC cells at 150 kHz, but significantly increased viability of AsPC-1 cells at all frequencies between 100 kHz and 300 kHz and that of BxPC-3 and BxGem cells at 250 kHz. Apoptosis or necrosis were not induced. Non-malignant CRL-4023 cells were not affected at 150 kHz. TTFields damaged PDAC cell lines but even favored their viability at very weak field strength and unfavorable frequency or inadequate field direction.

Sulforaphane Inhibits the Expression of Long Noncoding RNA H19 and Its Target APOBEC3G and Thereby Pancreatic Cancer Progression.

Pancreatic ductal adenocarcinoma (PDAC) is extremely malignant and the therapeutic options available usually have little impact on survival. Great hope is placed on new therapeutic targets, including long noncoding RNAs (lncRNAs), and on the development of new drugs, based on e.g., broccoli-derived sulforaphane, which meanwhile has shown promise in pilot studies in patients. We examined whether sulforaphane interferes with lncRNA signaling and analyzed five PDAC and two nonmalignant cell lines, patient tissues (n = 30), and online patient data (n = 350). RT-qPCR, Western blotting, MTT, colony formation, transwell and wound healing assays; gene array analysis; bioinformatics; in situ hybridization; immunohistochemistry and xenotransplantation were used. Sulforaphane regulated the expression of all of five examined lncRNAs, but basal expression, biological function and inhibition of H19 were of highest significance. H19 siRNA prevented colony formation, migration, invasion and Smad2 phosphorylation. We identified 103 common sulforaphane- and H19-related target genes and focused to the virus-induced tumor promoter APOBEC3G. APOBEC3G siRNA mimicked the previously observed H19 and sulforaphane effects. In vivo, sulforaphane- or H19 or APOBEC3G siRNAs led to significantly smaller tumor xenografts with reduced expression of Ki67, APOBEC3G and phospho-Smad2. Together, we identified APOBEC3G as H19 target, and both are inhibited by sulforaphane in prevention of PDAC progression.

Novel plant microRNAs from broccoletti sprouts do not show cross-kingdom regulation of pancreatic cancer.

Food-derived plant microRNAs are suggested to control human genes by "cross-kingdom" regulation. We examined microRNAs in sprouts from Brassica rapa sylvestris, known as broccoletti, which are widely used as sulforaphane supplements, and assessed their influence on pancreatic cancer. RNA was isolated from 4-day-old sprouts, followed by deep sequencing and bioinformatic analysis. We identified 2 new and 745 known plant microRNA sequences in the miRbase database and predicted 15,494 human target genes and 76,747 putative 3'-UTR binding sites in these target genes. The most promising candidates were the already known microRNA sequence bra-miR156g-5p and the new sequence Myseq-330, both with predicted human target genes related to apoptosis. The overexpression of the respective oligonucleotides by lipofection did not alter the viability, apoptosis, clonogenicity, migration or associated protein expression patterns in pancreatic cancer cells. These data demonstrate that broccoletti sprouts contain microRNA sequences with putative binding sites in human genes, but the sequences evaluated here did not affect cancer growth. Our database of broccoletti-derived microRNA sequences provides a valuable tool for future analysis.

Inhibition of miR30a-3p by sulforaphane enhances gap junction intercellular communication in pancreatic cancer.

The therapy resistance of pancreatic cancer is associated with the loss of gap junction intercellular communication and connexin 43 expression. The broccoli isothiocyanate sulforaphane restored these features and therapy sensitivity. We investigated whether microRNA signaling is involved. Established cell lines and a patient tissue array (n = 96) were evaluated by miRNA and gene array, bioinformatics, expression studies, in situ hybridization and immunohistochemistry. Sulforaphane inhibited the expression of our top candidate miR30a-3p. Upon transfection of miR30a-3p inhibitors, the gemcitabine bystander effect, Cx43 expression and intercellular communication increased, whereas miR30a-3p mimics inhibited the luciferase activity of a Cx43 3'-UTR construct. miR30a-3p-overexpressing tumor xenografts had a decreased tumor volume and increased gemcitabine sensitivity. In patient tissues, a higher expression of miR30a-3p and a lower expression of Cx43 correlated with malignancy. These findings provide new knowledge and suggest sulforaphane as cotreatment for pancreatic cancer.

Sulforaphane Induces miR135b-5p and Its Target Gene, RASAL2, thereby Inhibiting the Progression of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal tumors, with poor therapeutic options in the advanced state. The broccoli-derived anti-inflammatory agent sulforaphane was shown to inhibit the progression of pancreatic cancer and other tumor entities. We examined the involvement of pancreatic cancer cell lines were evaluated by microRNA and gene expression arrays, bioinformatics, in silico analysis, qRT-PCR, western blotting, immunohistochemistry, in situ hybridization, self-renewal and differentiation assays, and in vivo xenograft studies. We selected the top nine differentially expressed microRNAs, and miR135b-5p was chosen as the most important candidate for the sulforaphane-induced upregulation of the tumor suppressor gene RASAL2. The expression of miR135b-5p and RASAL2 was almost absent in malignant pancreatic tissues and cell lines, but not in their normal counterparts. Lipofection of miR135b-5p enhanced RASAL2 expression and inhibited ERK1/2 signaling, viability, self-renewal capacity, and tumor growth. These results indicate that miR135b-5p acts as a tumor suppressor via the induction of RASAL2 in PDA.

MicroRNA-365a-3p inhibits c-Rel-mediated NF-κB signaling and the progression of pancreatic cancer.

NF-κB contributes to the aggressiveness of pancreatic ductal adenocarcinoma (PDA), which is counteracted by the bioactive agent sulforaphane. We investigated sulforaphane-induced microRNA signaling and its influence on progression features. Using established cell lines, microRNA and gene arrays, we predicted miR-365a as the top candidate for the sulforaphane-induced inhibition of the NF-κB subunit c-Rel. The lipofection of miR-365a-3p mimics inhibited the luciferase activity of a c-Rel 3'-UTR construct, as well as c-Rel expression, NF-κB activity, and tumor viability, migration, and clonogenicity, whereas apoptosis was induced. In vivo, miR-365a-3p reduced the volume of tumor xenografts and the expression of progression markers. In a tissue array, the expression of miR-365a-3p was absent in almost all 91 malignant tissues but not in 5 normal tissues, thus confirming the previous results. Our observations suggest that sulforaphane-induced miR-365a-3p expression inhibits NF-κB activity by downregulating c-Rel, which prevents the progression of PDA.

Dexamethasone-induced inhibition of miR-132 via methylation promotes TGF-ß-driven progression of pancreatic cancer.

Glucocorticoids (GCs) such as dexamethasone (DEX) are administered as cancer co­treatment for palliative purposes due to their pro­apoptotic effects in lymphoid cancer and limited side effects associated with cancer growth and chemotherapy. However, there is emerging evidence that GCs induce therapy resistance in most epithelial tumors. Our recent data reveal that DEX promotes the progression of pancreatic ductal adenocarcinoma (PDA). In the present study, we examined 1 primary and 2 established PDA cell lines, and 35 PDA tissues from patients who had received (n=14) or not received (n=21) GCs prior to surgery. Through microRNA microarray analysis, in silico, and RT­qPCR analyses, we identified 268 microRNAs differentially expressed between DEX­treated and untreated cells. With a focus on cancer progression, we selected miR­132 and its target gene, transforming growth factor-ß2 (TGF­ß2), as top candidates. miR­132 mimics directly bound to the 3' untranslated region (3'UTR) of a TGF­ß2 luciferase construct and enhanced expression, as shown by increased luciferase activity. By contrast, DEX inhibited miR­132 expression via promoter methylation. miR­132 mimics also reduced DEX­induced clonogenicity, migration and expression of vimentin and E­cadherin in vitro and in tumor xenografts. In patients, GC intake prior to surgery enhanced global hypermethylation and expression of TGF­ß2 in tissues; expression of miR­132 was detected but could not be quantified. Our results demonstrate that DEX­mediated inhibition of miR­132 is a key mediator in the progression of pancreatic cancer, and the findings provide a foundation for miRNA­based therapies.

Simvastatin inhibits sonic hedgehog signaling and stemness features of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) has poor therapeutic options. Recent patient studies indicate that cholesterol-lowering statins have anti-tumor capacities. We examined several established and primary PDA and normal cell lines as well as PDA patient tissues (n = 68). We found that simvastatin inhibited viability, stemness, tumor growth and metastasis and that it enhanced the efficacy of gemcitabine. These changes were associated with modulation of Shh-related gene expression. Overexpression of Shh prevented the anti-cancer effect of simvastatin, and inhibition of Shh mimicked the simvastatin effect. In PDA tissues, expression levels of Shh, downstream mediators of Shh and progression markers, namely, cMet, CxCR4 and Vimentin, were lower when patients were prescribed statin medication prior to surgery. These results suggested that statins are cost effective and well-tolerated drugs for prevention and co-treatment of PDA.

Intraductal papillary mucinous neoplasm of the pancreas rapidly xenografts in chicken eggs and predicts aggressiveness.

Intraductal papillary mucinous neoplasm (IPMN) of the pancreas has a high risk of progressing to invasive pancreatic ductal adenocarcinoma (PDA), but experimental models for IPMN are largely missing. New experimental systems for the molecular characterization of IPMN and for personalized prognosis and treatment options for IPMN are urgently needed. We analyzed the potential use of fertilized chicken eggs for the culture of freshly resected IPMN tissue. We transplanted 49 freshly resected IPMN tissues into eggs and compared the growth characteristics to IPMN tissues transplanted into mice; this was followed by an analysis of histology, morphology, and marker expression. Of the IPMN tissues transplanted into eggs, 63% formed tumor xenografts within 4 days, while none of the 12 IPMN tissues transplanted into immunodeficient mice engrafted. In the eggs, the grafting efficiency of high-grade (n = 14) and intermediate-grade (n = 17) dysplasia was 77% and was significantly higher than the 39% grafting efficiency of low-grade dysplasia (n = 18). According to mucinous expression, 46 IPMN tissues were classified into gastric (n = 6), intestinal (n = 3), oncocytic (n = 23), and pancreatobiliary (n = 14) subtypes. The grafting efficiency was highest for the pancreatobiliary subtype (86%), followed by the oncocytic (70%), gastric (33%) and intestinal (33%) subtypes. The morphology and expression patterns of mucins, progression markers and pancreatic ductal markers were comparable between the primary IPMN tissues and their xenograft copies. The individual tumor environment was largely maintained during subtransplantation, as evaluated upon passage 6. This new IPMN model may facilitate experimental studies and treatment decisions for the optimal personalized management of IPMN.

Dexamethasone mediates pancreatic cancer progression by glucocorticoid receptor, TGFß and JNK/AP-1.

Glucocorticoids such as dexamethasone are widely co-prescribed with cytotoxic therapy because of their proapoptotic effects in lymphoid cancer, reduction of inflammation and edema and additional benefits. Concerns about glucocorticoid-induced therapy resistance, enhanced metastasis and reduced survival of patients are largely not considered. We analyzed dexamethasone-induced tumor progression in three established and one primary human pancreatic ductal adenocarcinoma (PDA) cell lines and in PDA tissue from patients and xenografts by FACS and western blot analysis, immunohistochemistry, MTT and wound assay, colony and spheroid formation, EMSA and in vivo tumor growth and metastasis of tumor xenografts on chicken eggs and mice. Dexamethasone in concentrations observed in plasma of patients favored epithelial-mesenchymal transition, self-renewal potential and cancer progression. Ras/JNK signaling, enhanced expression of TGFß, vimentin, Notch-1 and SOX-2 and the inhibition of E-cadherin occurred. This was confirmed in patient and xenograft tissue, where dexamethasone induced tumor proliferation, gemcitabine resistance and metastasis. Inhibition of each TGFß receptor-I, glucocorticoid receptor or JNK signaling partially reversed the dexamethasone-mediated effects, suggesting a complex signaling network. These data reveal that dexamethasone mediates progression by membrane effects and binding to glucocorticoid receptor.

microRNA-210 overexpression inhibits tumor growth and potentially reverses gemcitabine resistance in pancreatic cancer.

Resistance to first-line chemotherapies like gemcitabine contributes to high disease lethality in pancreatic cancer. By microarray and qRT-PCR, we observed significant downregulation of microRNA-210 in gemcitabine-resistant cells. The overexpression of microRNA-210 was toxic to gemcitabine-resistant cells and enhanced gemcitabine sensitivity. MicroRNA-210 overexpression induced caspase-3-mediated apoptosis, and inhibited colony formation. Computationally, ABCC5, a highly expressed gene in our array data, was identified as a potential target of microRNA-210 and the overexpression of ABCC5 in gemcitabine-resistant cells was confirmed by qRT-PCR. MicroRNA-210 overexpression reduced ABCC5 mRNA levels and inhibited a luciferase reporter expressing the ABCC5 3' UTR. The expression pattern of microRNA-210 and ABCC5 was mirrored in all of 5 pancreatic cancer cell lines used. Likewise, microRNA-210 transfection nearly totally inhibited tumor xenograft growth, proliferation and metastasis without obvious side effects in vivo. Also, an absence or low expression of microRNA-210 correlated to high ABCC5 expression in the majority of malignant patient tissues from a total of 101 patient tissues examined. Our observations provide at first glance, an important function for microRNA-210 in regulation of gemcitabine responsiveness by it's target gene ABCC5.

Up-regulation of microRNA let-7c by quercetin inhibits pancreatic cancer progression by activation of Numbl.

Pancreatic Ductal Adenocarcinoma (PDA) is a highly malignant tumor with poor prognosis. MicroRNAs (miRs) may offer novel therapeutic approaches to treatment. The polyphenol quercetin, present in many fruits and vegetables, possesses anti-carcinogenic properties. To unravel the effect of quercetin to miR signaling we performed miR profiling in PDA cells before and after quercetin treatment, followed by biostatistical analysis. miR let-7c was among the top up-regulated candidates after quercetin treatment, as measured by qRT-PCR and confirmed in two established and one primary PDA cell lines. By computational analysis we identified the Notch-inhibitor Numbl as let-7c target gene. This was strengthened by luciferase assays, where lipofected let-7c mimics induced a Numbl 3-UTR wild type construct, but not the mutated counterpart. Let-7c induced Numbl mRNA and protein expression but inhibited Notch just like quercetin. It also inhibited colony formation, wound healing, and protein expression of progression markers. In vivo xenotransplantation of PDA cells and subsequent intravenous injection of let-7c resulted in a significant decrease in tumor mass without obvious toxic effects in the fertilized chick egg model. The delivery rate of the miR mimics to the tumor mass was 80%, whereas minor amounts were present in host tissue. By immunohistochemistry we demonstrated that let-7c inhibited Notch and progression markers but up-regulated Numbl. These findings show that quercetin-induced let-7c decreases tumor growth by posttranscriptional activation of Numbl and indirect inhibition of Notch.