Pancreatic Carcinoma Cell Lines Research Articles

Derivation of pancreatic acinar cell carcinoma cell line HS-1 as a patient-derived tumor organoid.

Acinar cell carcinoma (ACC) of the pancreas is a malignant tumor of the exocrine cell lineage with a poor prognosis. Due to its rare incidence and technical difficulties, few authentic human cell lines are currently available, hampering detailed investigations of ACC. Therefore, we applied the organoid culture technique to various types of specimens, such as bile, biopsy, and resected tumor, obtained from a single ACC patient. Despite the initial propagation, none of these organoids achieved long-term proliferation or tolerated cryopreservation, confirming the challenging nature of establishing ACC cell lines. Nevertheless, the biopsy-derived early passage organoid developed subcutaneous tumors in immunodeficient mice. The xenograft tumor histologically resembled the original tumor and gave rise to infinitely propagating organoids with solid features and high levels of trypsin secretion. Moreover, the organoid stained positive for carboxylic ester hydrolase, a specific ACC marker, but negative for the duct cell marker CD133 and the endocrine lineage marker synaptophysin. Hence, we concluded the derivation of a novel ACC cell line of the pure exocrine lineage, designated HS-1. Genomic analysis revealed extensive copy number alterations and mutations in EP400 in the original tumor, which were enriched in primary organoids. HS-1 displayed homozygous deletion of CDKN2A, which might underlie xenograft formation from organoids. Although resistant to standard cytotoxic agents, the cell line was highly sensitive to the proteasome inhibitor bortezomib, as revealed by an in vitro drug screen and in vivo validation. In summary, we document a novel ACC cell line, which could be useful for ACC studies in the future.

The Effect of 1,2,4-Triazole-3-thiol Derivatives Bearing Hydrazone Moiety on Cancer Cell Migration and Growth of Melanoma, Breast, and Pancreatic Cancer Spheroids.

4-Phenyl-3-[2-(phenylamino)ethyl]-1H-1,2,4-triazole-5(4H)-thione was used as a starting compound for the synthesis of the corresponding 1,2,4-triazol-3-ylthioacetohydrazide, which reacts with isatins and various aldehydes bearing aromatic and heterocyclic moieties provided target hydrazones. Their cytotoxicity was tested by the MTT assay against human melanoma IGR39, human triple-negative breast cancer (MDA-MB-231), and pancreatic carcinoma (Panc-1) cell lines. The selectivity of compounds towards cancer cells was also studied. In general, the synthesized compounds were more cytotoxic against the melanoma cell line. N′-(2-oxoindolin-3-ylidene)-2-((4-phenyl-5-(2-(phenylamino)ethyl)-4H-1,2,4-triazol-3-yl)thio)acetohydrazide, N′-((1H-pyrrol-2-yl)methylene)-2-((4-phenyl-5-(2-(phenylamino)ethyl)-4H-1,2,4-triazol-3-yl)thio)acetohydrazide and N′-(2-hydroxy-5-nitrobenzylidene)-2-((4-phenyl-5-(2-(phenylamino)ethyl)-4H-1,2,4-triazol-3-yl)thio)acetohydrazide were identified as the most active among all synthesized compounds in 3D cell cultures. N′-(4-(dimethylamino)benzylidene)-2-((4-phenyl-5-(2-(phenylamino)ethyl)-4H-1,2,4-triazol-3-yl)thio)acetohydrazide inhibited all cancer cell migration, was characterized as relatively more selective towards cancer cells, and could be further tested as an antimetastatic candidate.

Metabolites from the plant endophytic fungus Penicillium sp. CPCC 401423 and their cytotoxic activity against MIA PaCa-2 cells

Twenty-two metabolites were isolated from Penicillium sp. CPCC 401423 cultured on rice. The structures of all compounds were elucidated mainly by MS and NMR analysis as well as the necessary CD experimental evidence, of which penicillidione A (1), penicillidione B (2), (E)-4-[(4-acetoxy-3-methyl-2-butenyl)oxy]phenylacetic acid (3), (S)-2-hydroxy-2-{4-[(3-methyl-2-butenyl)oxy]phenyl} (4), (S)-4-(2,3-dihydroxy-3-methyl-butoxy)phenylacetic acid (5), (E)-4-[(3-carboxy-2-butenyl)oxy]benzoic acid (6), (Z)-4-[(4-hydroxy-3-methyl-2-butenyl)oxy]benzoic acid (7), open-cycled N-demethylmelearoride A (12), and penostatin M (16) were identified as new compounds. The cytotoxic activity against human pancreatic carcinoma cell line MIA PaCa-2a was detected. Among them, compounds 13–15 and 22 displayed significant cytotoxicity against MIA-PaCa-2 cells with IC50 values of 8.9, 36.5, 31.8, and 22.3 µM, respectively (positive control gemcitabine IC50 65.0 µM).

Gemcitabine Resistance in Pancreatic Ductal Carcinoma Cell Lines Stems from Reprogramming of Energy Metabolism

Pancreatic ductal adenocarcinoma (PDAC) is associated with poor prognosis because it is often detected at an advanced stage, and drug resistance interferes with treatment. However, the mechanism underlying drug resistance in PDAC remains unclear. Here, we investigated metabolic changes between a parental PDAC cell line and a gemcitabine (GEM)-resistant PDAC cell line. We established a GEM-resistant cell line, MIA-G, from MIA-PaCa-2 parental (MIA-P) cells using continuous therapeutic-dose GEM treatment. MIA-G cells were also more resistant to 5-fluorouracil in comparison to MIA-P cells. Metabolic flux analysis showed a higher oxygen consumption rate (OCR) in MIA-G cells than in MIA-P cells. Notably, OCR was suppressed by GEM treatment only in MIA-G cells. GEM treatment increased mitochondrial membrane potential and mitochondrial reactive oxygen species (ROS) in MIA-P cells, but not in MIA-G cells. Glutamine uptake and peroxidase levels were elevated in MIA-G cells. The antioxidants N-acetyl-L-cysteine and vitamin C increased the sensitivity to GEM in both cell lines. In MIA-G cells, the expression of the mitochondrial transcription factor A also decreased. Furthermore, rotenone reduced the sensitivity of MIA-P cells to GEM. These findings suggest that the suppression of oxidative phosphorylation contributes to GEM resistance by reducing ROS production. Our study provides a new approach for reducing GEM resistance in PDAC.

Lipocalin-2 inhibits pancreatic cancer stemness via the AKT/c-Jun pathway.

Cancerstemcells(CSCs) are involved incancerrecurrenceandmetastasisowing to their self-renewal properties and drug-resistance capacity. Lipocalin-2 (Lcn2) of the lipocalin superfamily is highly expressed in pancreatic cancer. Nevertheless, reports on the involvement of Lcn2 in the regulation of pancreatic CSC properties are scant. This study is purposed to investigate whether Lcn2 plays a crucial role in CSC renewal and stemness maintenance in pancreatic carcinoma. Immunohistochemistry results of tumor tissue chips together with Gene Expression Omnibus sequencing files confirmed that Lcn2 is highly expressed in pancreatic carcinoma compared with that in normal tissues. The exogenous expression of Lcn2 attenuated CSC-associated SOX2, CD44, and EpCAM expression and suppressed sarcosphere formation and tumorigenesis in the pancreatic carcinoma cell line PANC-1, which showed low expression of Lcn2. However, Lcn2 knockout in BxPC-3 cell line, which presented high Lcn2 expression, promoted CSC stemness, further enhancing sarcosphere formation and tumorigenesis. Moreover, Lcn2 was found to regulate stemness in pancreatic cancer depending on the activation of AKT and c-Jun. Lcn2 suppresses stemness properties in pancreatic carcinoma by activating the AKT-c-Jun pathway, and thus, it may be a novel candidate to suppress the stemness of pancreatic cancer. This study provides a new insight into disease progression.

Hetaryl- and heteroarylvinyl-substituted nitrofurans identified as non-cytotoxic selective antitubercular agents

New (E)-5-[2-(5-nitrofuran-2-yl)vinyl]-1,2,4-oxadiazoles and 5-(5-nitrofuran-2-yl)-1,2,4-oxadiazoles were synthesized via the base-promoted condensation of nitrofuran-containing acyl chlorides with amidoximes. Testing these compounds against Gram-negative E. coli, Gram-positive B. subtilis and S. aureus as well as M. tuberculosis HRv37 strain revealed three compounds being selectively antimycobacterial. None of these compounds displayed any cytotoxicity towards human pancreatic epithelioid carcinoma cell line, PANC-1.

Targeting Tumor Cells Overexpressing the Human Epidermal Growth Factor Receptor 3 with Potent Drug Conjugates Based on Affibody Molecules.

Increasing evidence suggests that therapy targeting the human epidermal growth factor receptor 3 (HER3) could be a viable route for targeted cancer therapy. Here, we studied a novel drug conjugate, ZHER3-ABD-mcDM1, consisting of a HER3-targeting affibody molecule, coupled to the cytotoxic tubulin polymerization inhibitor DM1, and an albumin-binding domain for in vivo half-life extension. ZHER3-ABD-mcDM1 showed a strong affinity to the extracellular domain of HER3 (KD 6 nM), and an even stronger affinity (KD 0.2 nM) to the HER3-overexpressing pancreatic carcinoma cell line, BxPC-3. The drug conjugate showed a potent cytotoxic effect on BxPC-3 cells with an IC50 value of 7 nM. Evaluation of a radiolabeled version, [99mTc]Tc-ZHER3-ABD-mcDM1, showed a relatively high rate of internalization, with a 27% internalized fraction after 8 h. Further in vivo evaluation showed that it could target BxPC-3 (pancreatic carcinoma) and DU145 (prostate carcinoma) xenografts in mice, with an uptake peaking at 6.3 ± 0.4% IA/g at 6 h post-injection for the BxPC-3 xenografts. The general biodistribution showed uptake in the liver, lung, salivary gland, stomach, and small intestine, organs known to express murine ErbB3 naturally. The results from the study show that ZHER3-ABD-mcDM1 is a highly potent and selective drug conjugate with the ability to specifically target HER3 overexpressing cells. Further pre-clinical and clinical development is discussed.

Sunitinib potentiates the cytotoxic effect of electrochemotherapy in pancreatic carcinoma cells.

BackgroundOne of the new treatment options for unresectable locally advanced pancreatic cancer is electrochemotherapy (ECT), a local ablative therapy that potentiates the entry of chemotherapeutic drugs into the cells, by the application of an electric field to the tumor. Its feasibility and safety were demonstrated in preclinical and clinical studies; however, there is a lack of preclinical studies assessing the actions of different drugs used in ECT, their mechanisms and interactions with other target drugs that are used in clinical practice.Materials and methodsThe aim of the study was to determine the cytotoxicity of two chemotherapeutic drugs usually used in ECT (bleomycin and cisplatin) in the BxPC-3 human pancreatic carcinoma cell line and evaluate the interactions of ECT with the targeted drug sunitinib. First, the cytotoxicity of ECT using both chemotherapeutics was determined. In the next part, the interactions of ECT and sunitinib were evaluated through determination of combined cytotoxicity, sunitinib targets and kinetics of cell death.ResultsThe results demonstrate that ECT is effective in pancreatic cancer cell line, especially when bleomycin is used, with the onset of cell death in the first hours after the treatment, reaching a plateau at 20 hours after the treatment. Furthermore, we provide the rationale for combining ECT with bleomycin and the targeted drug sunitinib to potentiate cytotoxicity. The combined treatment of sunitinib and ECT was synergistic for bleomycin only at the highest used concentration of bleomycin 0.14 μM, whereas with lower doses of bleomycin, this effect was not observed. The interaction of ECT and treatment with sunitinib was confirmed by course of the cell death, also indicating on synergism.ConclusionsECT and sunitinib combined treatment has clinical potential, and further studies are warranted.

ZNF488 Promotes the Invasion and Migration of Pancreatic Carcinoma Cells through the Akt/mTOR Pathway.

Objective Studies have demonstrated that zinc finger protein 488 (ZNF488) is highly expressed in pancreatic carcinoma (PC), but its effect on PC and its molecular mechanism remains unclear. Methods Real-time fluorescent quantitative PCR (RT-qPCR) was employed to detect the ZNF488 expression in PC patients' cancer tissues and cell lines. After interfering with or overexpressing ZNF488 in PANC-1 and AsPC-1 cells, respectively, the CCK-8, cell cloning, Transwell, and scratch assays were performed to detect cell proliferation, cell viability, invasion ability, and migration ability. In addition, Western blot was applied to assess the protein expression of Akt, p-Akt, mTOR, and p-mTOR in the Akt-mTOR pathway. Results The ZNF488 expression was evidently raised in PC tissues and cell lines, and the starBase V3.0 database indicated that the higher the ZNF488 expression, the lower the survival rate of PC patients. Furthermore, we discovered that overexpressing ZNF488 can markedly promote the proliferation, invasion, and migration of PC cells. At the same time, highly expressed ZNF488 distinctly increased the p-Akt and p-mTOR expressions and the p-Akt/Akt and p-mTOR/mTOR ratios. However, after knocking down the ZNF488 expression, it had the opposite results. In addition, the Akt agonist SC79 can alleviate the effect of ZNF488 knockdown on Akt/mTOR pathway-related proteins, while Akt inhibitor AZD5363 had the opposite effect. Conclusion ZNF488 could promote the proliferation, invasion, and migration of PC cells, and its mechanism may be related to the activation of the Akt/mTOR pathway. This study demonstrated that ZNF488 could be used as a molecular target for diagnosing and treating PC.

Long non‑coding RNA CTBP1‑AS2 upregulates USP22 to promote pancreatic carcinoma progression by sponging miR‑141‑3p.

Long non-coding RNAs (lncRNAs) feature prominently in pancreatic carcinoma progression. The present study aimed to clarify the biological functions, clinical significance and underlying mechanism of lncRNA CTBP1 antisense RNA 2 (CTBP1-AS2) in pancreatic carcinoma. Reverse transcription-quantitative PCR was performed to assess the expression levels of CTBP1-AS2, microRNA (miR)-141-3p and ubiquitin-specific protease 22 (USP22) mRNA in pancreatic carcinoma tissues and cell lines. Western blotting was used to examine USP22 protein expression in pancreatic carcinoma cell lines. Loss-of-function experiments were used to analyze the regulatory effects of CTBP1-AS2 on proliferation, apoptosis, migration and invasion of pancreatic carcinoma cells. Dual-luciferase reporter assay was used to examine the binding relationship between CTBP1-AS2 and miR-141-3p, as well as between miR-141-3p and USP22. It was demonstrated that CTBP1-AS2 expression was markedly increased in pancreatic carcinoma tissues and cell lines. High CTBP1-AS2 expression was associated with advanced clinical stage and lymph node metastasis of patients. Functional experiments confirmed that knocking down CTBP1-AS2 significantly inhibited pancreatic carcinoma cell proliferation, migration and invasion, and promoted cell apoptosis. In terms of mechanism, it was found that CTBP1-AS2 adsorbed miR-141-3p as a molecular sponge to upregulate the expression level of USP22. In conclusion, lncRNA CTBP1-AS2 may be involved in pancreatic carcinoma progression by regulating miR-141-3p and USP22 expressions; in addition, CTBP1-AS2 may be a diagnostic biomarker and treatment target for pancreatic carcinoma.

Изучение влияния детонационных наноалмазов и их конъюгатов с доксорубицином и диоксадэтом на функцию митохондрий

Targeted drug delivery is aimed at preventing drugs’ nonspecific distribution in different tissues. One of efficient ways for drug’s targeted delivery is using denonation nanodiamonds. These nanoparticles are an advantageous material for nanomedicine due to their low cost, biocompatibility, hardness and chemical stability. Majority of blood plasma proteins and blood cell surfaces are charged negatively. Therefore biocompatibility and long circulation lifetimes of carboxylated nanodiamonds can be partially attributed to their negative surface charge. The presence of different functional groups on nanodiamond surfaces enables their functionalization with a variety of therapeutic agents, including antitumour drugs. One of them is dioxadet, a derivative of symmetric triazine. It is becoming evident that mitochondria play crucial role in metastasis and chemoresistance of tumors, which makes them a very promising target for anticancer therapy. In particular, proton translocating F1Fo-ATPase/ATP synthase can be one of these targets. This enzyme is responsible for making the majority of ATP in the cell by oxidative phosphorylation. More than 300 natural and synthesized compounds are currently known to interact with or inhibit F1Fo, but a comparative study of their effects on energy metabolism has only recently started. In this work we estimated effects of the cytostatic drugs doxorubicin, dioxadet and their DND conjugates on the activity of F1Fo ATPase as well as on the membrane potential. Two model systems were employed: 1) E. coli F1Fo-ATPase reconstituted into phosphatidylcholine liposomes; 2) Pancreatic carcinoma cell line PANC1. It was found that the nanodiamonds and conjugates exhibited the same uncoupling effect, partially dissipating the proton gradient across the membrane of proteoliposomes containing F1Fo-ATPase. In addition, the nanodiamonds inhibited ATPase activity. In an experiment on the effect on the mitochondrial membrane potential (PANC 1 cell line), conjugates of nanodiamonds with doxorubicin demonstrated a more pronounced effect compared to doxorubicin, which may be due to a combination of the uncoupling effect of nanodiamonds and doxorubicin-dependent generation of reactive oxygen species. The data reported in this paper shed light on effect of the carboxylated nanodiamonds on the cellular bioenergetics. These findings are crucial for further development of nanodiamond-based targeted drug delivery systems, in particular decreasing their toxicity by selecting optimal strategies of nanodiamond synthesis as well as drug immobilization on their surfaces. The use of artificial models of natural membranes, along with cell cultures, is necessary for a deeper and more accurate understanding of the mechanism of action of cytostatics.

TCF12 activates MAGT1 expression to regulate the malignant progression of pancreatic carcinoma cells.

As a highly malignant gastrointestinal tumor, pancreatic carcinoma (PC) has poor prognosis due to its low early diagnosis rate, advanced tumor resection and chemotherapy resistance. Magnesium transporter 1 (MAGT1) is a magnesium ion transporter located on the cell membrane, which shows promotive effects on biological behaviors of multiple tumor cells. The aim of the present study was to investigate the role of MAGT1 in the progression of PC and its potential molecular mechanism. Based on the Gene Expression Profiling Interactive Analysis website, MAGT1 was highly expressed in tissues from patients with PC and was associated with poor prognosis. In functional experiments, MAGT1 was highly expressed in PC cell lines. The Cell Counting Kit-8, gap closure and Transwell assays, and western blot analysis, were used to investigate the effects of MAGT1 overexpression or knockdown on the biological behaviors of PC cells. It was found that MAGT1 promoted the proliferation, migration and invasion of PC cells in vitro. According to the Encyclopedia of RNA Interactomes website, transcription factor 12 (TCF12) mRNA expression level was positively correlated with MAGT1 expression level in the tissues from patients with PC. Positive targeting regulation of MAGT1 by TCF12 was also confirmed using a dual-luciferase gene reporter assay and chromatin immunoprecipitation. In addition, knockdown of TCF12 expression inhibited the proliferation and migration of PC cells, but overexpression of MAGT1 expression partly reversed this. These results suggested that TCF12 could promote the proliferation, migration and invasion of PC cells by activating MAGT1 expression, which was associated with poor prognosis. These findings suggest that MAGT1 could be a promising biomarker for the occurrence, progression and prognosis of PC.

NS-11021 Modulates Cancer-Associated Processes Independently of BK Channels in Melanoma and Pancreatic Duct Adenocarcinoma Cell Lines.

Simple SummaryPotassium channels permit the selective passage of K+ ions across the cell me brane and are important for setting the membrane potential and for the transmission of electrical signals in all cells. Ca2+ activated K+ channels provide a means to couple intracellular calcium signaling to changes of the membrane potential. Among these, the large-conductance Ca2+-activated K+ channel, known as BK, has been proposed to be involved in several cancer-associated processes. In the present work, we tested various BK channel activators for anti-cancer effects in melanoma and pancreatic duct carcinoma cell lines. Only one of the activators (NS-11021) had effects on cancer-associated processes. However, the compound, as a side-effect, also increased the intracellular Ca2+ concentration independently of BK channel activation. Overall, we conclude that the activation of the BK channel by itself is not sufficient to produce beneficial anti-cancer effects.Potassium channels have emerged as regulators of carcinogenesis, thus introducing possible new therapeutic strategies in the fight against cancer. In particular, the large-conductance Ca2+-activated K+ channel, often referred to as BK channel, is involved in several cancer-associated processes. Here, we investigated the effects of different BK activators, NS-11021, NS-19504, and BMS-191011, in IGR39 (primary melanoma cell line) and Panc-1 (primary pancreatic duct carcinoma cell line), highly expressing the channel, and in IGR37 (metastatic melanoma cell line) that barely express BK. Our data showed that NS-11021 and NS-19504 potently activated BK channels in IGR39 and Panc-1 cells, while no effect on channel activation was detected in IGR37 cells. On the contrary, BK channel activator BMS-191011 was less effective. However, only NS-11021 showed significant effects in cancer-associated processes, such as cell survival, migration, and proliferation in these cancer cell lines. Moreover, NS-11021 led to an increase of intracellular Ca2+ concentration, independent of BK channel activation, thus complicating any interpretation of its role in the regulation of cancer-associated mechanisms. Overall, we conclude that the activation of the BK channel by itself is not sufficient to produce beneficial anti-cancer effects in the melanoma and PDAC cell lines examined. Importantly, our results raise an alarm flag regarding the use of presumably specific BK channel openers as anti-cancer agents.

Phycocyanin inhibits pancreatic cancer metastasis via suppressing epithelial-mesenchymal transition and targeting Akt/β-catenin pathway.

According to our previous research, phycocyanin (PC) could inhibit pancreatic tumor growth obviously. However, little is known about the effects of phycocyanin on pancreatic tumor metastasis. This study was aimed to investigate whether phycocyanin inhibits pancreatic cancer cells' metastasis and the potential underlying mechanisms. Human pancreatic carcinoma cell lines PANC-1 and BxPC3 were treated with phycocyanin (5, 10, 20, and 40 μM) for 48 h or 72 h. BALB/c nude mice were inoculated intravenously with luciferase-PANC-1 cells to establish a tumor metastasis model. BALB/c nude mice were also inoculated subcutaneously with PANC-1 cells to establish a xenograft tumor model. Our results indicated that (1) phycocyanin inhibited migration, invasion, and movement of pancreatic cancer cells in vitro, and tumor metastasis in mouse xenograft tumor model; (2) phycocyanin significantly changed cell morphology and epithelial-mesenchymal transition (EMT) phenotype in pancreatic cancer cells; (3) phycocyanin decreased the phosphorylation level of Akt and the level of nuclear β-catenin. Moreover, administration of Akt activator SC79 counteracted the inhibitory effect of phycocyanin on the migration of pancreatic cancer cells, associated with the reversal of epithelial-mesenchymal transition and Akt/β-catenin signaling pathway. Taken together, our results suggest that phycocyanin inhibits pancreatic cancer metastasis via suppressing epithelial-mesenchymal transition and targeting the Akt/β-catenin pathway.

Calcium signaling induces a partial EMT.

Epithelial plasticity, or epithelial-to-mesenchymal transition (EMT), is a well-recognized form of cellular plasticity, which endows tumor cells with invasive properties and alters their sensitivity to various agents, thus representing a major challenge to cancer therapy. It is increasingly accepted that carcinoma cells exist along a continuum of hybrid epithelial-mesenchymal (E-M) states and that cells exhibiting such partial EMT (P-EMT) states have greater metastatic competence than those characterized by either extreme (E or M). We described recently a P-EMT program operating invivo by which carcinoma cells lose their epithelial state through post-translational programs. Here, we investigate the underlying mechanisms and report that prolonged calcium signaling induces a P-EMT characterized by the internalization of membrane-associated E-cadherin (ECAD) and other epithelial proteins as well as an increase in cellular migration and invasion. Signaling through Gαq-associated G-protein-coupled receptors (GPCRs) recapitulates these effects, which operate through the downstream activation of calmodulin-Camk2b signaling. These results implicate calcium signaling as a trigger for the acquisition of hybrid/partial epithelial-mesenchymal states in carcinoma cells.

CircCCT3 Acts as a Sponge of miR-613 to Promote Tumor Growth of Pancreatic Cancer Through Regulating VEGFA/VEGFR2 Signaling.

Background: Circular RNAs (CircRNAs) have been recently implicated in the progression of pancreatic cancer (PC).Aims: To investigate the involvement of CircCCT3 in PC and studying its interactions and functioning during the progression of PC in vitro and in vivo, using methods of molecular biology and bioinformatics.Study Design: Experimental study.Methods: The expressions of CircCCT3 and miR-613 in pancreatic ­carcinoma tissues and cell lines were evaluated by quantitative real-time polymerase chain reaction (PCR). The relationship between clinical pathologic features as well as the survival rate and CircCCT3 expression was analyzed with chi-square test and the Kaplan–Meier method. CCK-8, wound healing, transwell assays, and the fluorescein isothiocyanate-AnnexinV/propidium iodide (FITC-AnnexinV/PI) assay were used to assess cell proliferation, migration, invasion, and apoptosis after CircCCT3 overexpression or downregulation. The Dual-Luciferase reporter assay, RNA immunoprecipitation (RIP), RNA pull-down and fluorescence in situ hybridization (FISH) assays were performed to validate the potential interaction of CircCCT3, miR-613, and vascular endothelial growth factor (VEGFA). The nude mouse xenograft tumor assay was used to detect CircCCT3 effects on pancreatic tumorigenesis in vivo. Western blotting analysis was performed to examine the VEGFA and the vascular endothelial growth factor receptor 2 (VEGFR2) protein expressions following.Results: CircCCT3 expression was significantly increased in PC tissues (3.41 ± 0.57 vs. 1.00 ± 0.10, P < .01) and cell lines (Patu8988 2.57 ± 0.20; SW1990 2.88 ± 0.10; BxPC-3 2.45 ± 0.20; Panc02 2.99 ± 0.10 vs. H6c7 1.00 ± 0.10; all P < .001). CircCCT3 expression was negatively correlated with miR-613 expression. PC patients with high CircCCT3 expression exhibited significantly poorer overall survival rate than those patients with low CircCCT3 expression (P = .013). Moreover, it was found that CircCCT3 promoted cell proliferation, migration, and invasion, and inhibited cell apoptosis in PC cells. The CircCCT3 acted as a sponge for the miR-613 to facilitate VEGFA and VEGFR2 expression. si-CirCCT3 also inhibited tumor growth of PC in nude mice. si-CircCCT3 reduced VEGFA and VEGFR2 expression, whereas overexpression of CircCCT3 increased VEGFA and VEGFR2 expression.Conclusion: Increased CircCCT3 suggests a poor prognosis for PC patients and promotes the migration and invasion through targeting VEGFA/VEGFR2 signaling. CircCCT3 may serve as a potential and promising therapeutic target for PC treatment.

Cancer Cells Shuttle Extracellular Vesicles Containing Oncogenic Mutant p53 Proteins to the Tumor Microenvironment

Simple SummaryIn addition to the classical cell-to-cell communication patterns, extracellular vesicles (EVs) are instrumental in conveying molecular messages across cell types and have the potential to mediate changes at a tissue level. Since it is now appreciated that carcinomas are fundamentally reliant on two-way communication with activated cells in the tumor microenvironment, elucidating the roles of EVs exchange and of the cargo that is transferred is essential to obtain a thorough understanding of tumor progression. This study reveals that mutant p53 proteins—the result of the most frequent mutated gene in human cancer—are packed into EVs and delivered to neighboring cells with the potential to reprogram immune cells and subsequently establish a positive feedback loop that will enhance tumor progression. This non-cell autonomous role of mutant p53 is evidence of an extra layer of communication that is orchestrated by smaller vesicles that transfer oncogenic elements between cellular entities. Building on the foundation of our work on mutant p53, future studies may aim to characterize the potential activation of additional oncogenes, thus opening new paths of research at the interface of extracellular vesicles, cancer, and evolution.Extracellular vesicles (EVs) shed by cancer cells play a major role in mediating the transfer of molecular information by reprogramming the tumor microenvironment (TME). TP53 (encoding the p53 protein) is the most mutated gene across many cancer types. Mutations in TP53 not only result in the loss of its tumor-suppressive properties but also results in the acquisition of novel gain-of-functions (GOF) that promote the growth of cancer cells. Here, we demonstrate that GOF mutant p53 proteins can be transferred via EVs to neighboring cancer cells and to macrophages, thus modulating them to release tumor supportive cytokines. Our data from pancreatic, lung, and colon carcinoma cell lines demonstrate that the mutant p53 protein can be selectively sorted into EVs. More specifically, mutant p53 proteins in EVs can be taken up by neighboring cells and mutant p53 expression is found in non-tumor cells in both human cancers and in non-human tissues in human xenografts. Our findings shed light on the intricate methods in which specific GOF p53 mutants can promote oncogenic mechanisms by reprogramming and then recruiting non-cancerous elements for tumor progression.

Chlorophenyl-benzoxime inhibits pancreatic cancer cell proliferation, invasion and migration by down-regulating the expressions of interleukin-8 and cyclooxygenase-2

Purpose: To investigate the effects of chlorophenyl-benzoxime (CPBZX) on pancreatic cancer (PC) cell proliferation, invasion and migration, and the underlying mechanism of action.&#x0D; Methods: Pancreatic carcinoma cell lines (HuP-T4, HuP-T3 and BxPC-3) were cultured in Dulbecco's Modified Eagle medium (DMEM) containing 10 % fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (10 μg/mL) at 37 ˚C in a humidified atmosphere containing 5 % CO2 and 95 % air. Cell proliferation was assessed using MTT assay. Real-time quantitative polymerase chain reaction (qRTPCR) and Western blotting were employed for the determination of changes in the levels of expression of carcinoembryonic antigen (CEA), interleukin-8 (IL-8) and cyclooxygenase-2 (COX 2). Cell invasion and migration were determined using Transwell and wound healing assays, respectively.&#x0D; Results: The results of MTT assay showed that CPBZX significantly and dose-dependently inhibited the proliferation of PC cells (p &lt; 0.05). Incubation of HuP-T4 cells with CPBZX significantly and dosedependently reduced the invasive ability of the cells (p &lt; 0.05). The migratory ability of HuP-T4 cells was also significantly and dose-dependently inhibited by CPBZX (p &lt; 0.05). The results of Western blotting and qRT PCR showed that CPBZX treatment significantly and dose-dependently upregulated CEA mRNA expression (p &lt; 0.05). On the other hand, the expressions of IL-8 and COX-2 were significantly and dose-dependently down-regulated by CPBZX. Treatment of pancreatic tumor mice with CPBZX significantly decreased tumor growth and metastasis of tumor cells to the pulmonary tissues, liver and lymph nodes (p &lt; 0.05).&#x0D; Conclusion: The results of this study suggest that CPBZX inhibits the development and metastasis of PC via the down-regulation of IL-8 and COX 2 expressions, and therefore may find application in pancreatic cancer therapy.

Isoalantolactone inhibits pancreatic cancer proliferation by regulation of PI3K and Wnt signal pathway.

Background/aimsIsoalantolactone (IATL) is one of multiple isomeric sesquiterpene lactones and is isolated from inula helenium. IATL has multiple functions such as antibacterial, antihelminthic and antiproliferative activities. IATL also inhibits pancreatic cancer proliferation and induces apoptosis by increasing ROS production. However, the detailed mechanism of IATL-mediated pancreatic cancer apoptosis remains largely unknown.MethodsIn current study, pancreatic carcinoma cell lines (PANC-1, AsPC-1, BxPC-3) and a mouse xenograft model were used to determine the mechanism of IATL-mediated toxic effects.ResultsIATL (20μM) inhibited pancreatic adenocarcinoma cell lines proliferation in a time-dependent way; while scratch assay showed that IATL significantly inhibited PANC-1 scratch closure (P<0.05); Invasion assays indicated that IATL significantly attenuated pancreatic adenocarcinoma cell lines invasion on matrigel. Signal analysis showed that IATL inhibited pancreatic adenocarcinoma cell proliferation by blocking EGF-PI3K-Skp2-Akt signal axis. Moreover, IATL induced pancreatic adenocarcinoma cell apoptosis by increasing cytosolic Caspase3 and Box expression. This apoptosis was mediated by inhibition of canonical wnt signal pathway. Finally, xenograft studies showed that IATL also significantly inhibited pancreatic adenocarcinoma cell proliferation and induced pancreatic adenocarcinoma cell apoptosis in vivo.ConclusionsIATL inhibits pancreatic cancer proliferation and induces apoptosis on cellular and in vivo models. Signal pathway studies reveal that EGF-PI3K-Skp2-Akt signal axis and canonical wnt pathway are involved in IATL-mediated cellular proliferation inhibition and apoptosis. These studies indicate that IATL may provide a future potential therapy for pancreatic cancer.

MiR24-3p activity after delivery into pancreatic carcinoma cell lines exerts profound tumor-inhibitory effects through distinct pathways of apoptosis and autophagy induction.

Pancreatic cancer is among the most detrimental tumors, with novel treatment options urgently needed. The pathological downregulation of a miRNA in tumors can lead to the overexpression of oncogenes, thus suggesting miRNA replacement as novel strategy in cancer therapy. While the role of miR24 in cancer, including pancreatic carcinoma, has been described as ambiguous, it may hold great promise and deserves further studies. Here, we comprehensively analyze the effects of miR24-3p replacement in a set of pancreatic carcinoma cell lines. Transfection of miR24-3p mimics leads to profound cell inhibition in various 2D and 3D cell assays, based on the induction of apoptosis, autophagy and ROS. Comprehensive analyses of miR24-3p effects on the molecular level reveal the transcriptional regulation of several important oncogenes and oncogenic pathways. Based on these findings, miRNA replacement therapy was preclinically explored by treating tumor xenograft-bearing mice with miR24-3p mimics formulated in polymeric nanoparticles. The obtained tumor inhibition was associated with the induction of apoptosis and necrosis. Taken together, we identify miR24-3p as powerful tumor-inhibitory miRNA for replacement therapy, and describe a complex network of oncogenic pathways affected by miR24.