Comparative evaluation of methods for isolating small extracellular vesicles derived from pancreatic cancer cells

Abstract Background Small extracellular vesicles (sEVs) are nanosized vesicles involved in cell-to-cell communication. sEVs have been widely studied for clinical applications such as early detection of diseases and as therapeutics. Various methods for sEVs isolation are been using, but different methods may result in different qualities of sEVs and impact downstream analysis and applications. Here, we compared current isolation methods and performed a comparative analysis of sEVs from supernatant of cultured pancreatic cancer cells. Methods Ultracentrifugation, ultrafiltration and co-precipitation as concentration methods were firstly evaluated for yield, size, morphology and protein level of pellets. Then, isolate sEVs obtained by four different purification methods: size exclusion chromatography, density gradient ultracentrifugation, ultracentrifugation, and immunoaffinity capturing, were analysed and compared. Results For the concentration process, ultracentrifugation method obtained high quality and high concentration of pellets. For the purification process, immunoaffinity capturing method obtained the purest sEVs with less contaminants, while density gradient ultracentrifugation-based method obtained sEVs with the smallest size. Proteomic analysis revealed distinct protein contents of purified sEVs from different methods. Conclusions For isolating sEVs derived from supernatant of cultured pancreatic cancer cell line, ultracentrifugation-based method is recommended for concentration of sEVs, density gradient ultracentrifugation-based method may be applied for obtaining purified sEVs with controlled size, immunoaffinity capturing may be suitable for studies requiring sEVs with high purity but may loss subtypes of sEVs without specific protein marker.

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Comparative Evaluation of Methods for Isolating Small Extracellular Vesicles Derived from Pancreatic Cancer Cells

10.21203/rs.3.rs-110539/v2 ◽

2021 ◽

Author(s):

Jie-Min Wang ◽

Yong-Jiang Li ◽

Jun-Yong Wu ◽

Jia-Xin Cai ◽

Jing Wen ◽

...

Keyword(s):

Pancreatic Cancer ◽

Cancer Cells ◽

Density Gradient ◽

Extracellular Vesicles ◽

Pancreatic Cancer Cell Line ◽

Size Exclusion ◽

Density Gradient Ultracentrifugation ◽

High Concentration ◽

Protein Marker ◽

Pancreatic Cancer Cells

Abstract Background: Small extracellular vesicles (sEVs) are nanosized vesicles involved in cell-to-cell communication. sEVs have been widely studied for clinical applications such as early detection of diseases and as therapeutics. Various methods for sEVs isolation are been using, but different methods may result in different qualities of sEVs and impact downstream analysis and applications. Here, we compared current isolation methods and performed a comparative analysis of sEVs from supernatant of cultured pancreatic cancer cells.Methods: Ultracentrifugation, ultrafiltration and co-precipitation as concentration methods were firstly evaluated for yield, size, morphology and protein level of pellets. Then, isolate sEVs obtained by four different purification methods: size exclusion chromatography, density gradient ultracentrifugation, ultracentrifugation, and immunoaffinity capturing, were analysed and compared.Results: For the concentration process, ultracentrifugation method obtained high quality and high concentration of pellets. For the purification process, immunoaffinity capturing method obtained the purest sEVs with less contaminants, while density gradient ultracentrifugation-based method obtained sEVs with the smallest size. Proteomic analysis revealed distinct protein contents of purified sEVs from different methods. Conclusions: For isolating sEVs derived from supernatant of cultured pancreatic cancer cell line, ultracentrifugation-based method is recommended for concentration of sEVs, density gradient ultracentrifugation-based method may be applied for obtaining purified sEVs with controlled size, immunoaffinity capturing may be suitable for studies requiring sEVs with high purity but may loss subtypes of sEVs without specific protein marker.

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Comparative Evaluation of Methods for Isolating Small Extracellular Vesicles Derived from Pancreatic Cells

10.21203/rs.3.rs-110539/v1 ◽

2020 ◽

Author(s):

Jie-Min Wang ◽

Yong-Jiang Li ◽

Jun-Yong Wu ◽

Jia-Xin Cai ◽

Jing Wen ◽

...

Keyword(s):

Pancreatic Cancer ◽

Cancer Cells ◽

Density Gradient ◽

Extracellular Vesicles ◽

Cell Communication ◽

Size Exclusion ◽

Density Gradient Ultracentrifugation ◽

Pancreatic Cancer Cells ◽

Co Precipitation ◽

Therapeutic Study

Abstract Background: Small extracellular vesicles (sEVs) are nanosized vesicles involved in cell-to-cell communication. sEVs have been widely studied for clinical applications such as early detection of diseases and as therapeutics. Various methods for sEVs isolation have been using, but different methods may result in different qualities of sEVs and impact downstream analysis and applications. Here, we compared current isolation methods and performed a comparative analysis of sEVs derived from pancreatic cancer cells.Results: Ultracentrifugation, ultrafiltration and co-precipitation as concentration methods were firstly evaluated for yield, size, morphology and protein level of pellets. Then, isolate sEVs obtained by four different purification methods: size exclusion chromatography, density gradient ultracentrifugation, ultracentrifugation, and immunoaffinity capturing, were analysed and compared. For the concentration process, ultracentrifugation method obtained high quality and concentration pellets. For the purification process, immunoaffinity capturing method obtained the purest sEVs with less contaminants, while density gradient ultracentrifugation-based method obtained sEVs with the smallest size. Proteomic analysis revealed distinct protein contents of purified sEVs. Conclusions: For isolating sEVs derived from pancreatic cancer cells, ultracentrifugation-based method is recommended for concentration of sEVs, density gradient ultracentrifugation-based method may be suitable for isolation of sEVs for therapeutic study, immunoaffinity capturing may be applied for studies exploring sEVs as biomarkers.

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Cyathus striatus Extract Induces Apoptosis in Human Pancreatic Cancer Cells and Inhibits Xenograft Tumor Growth In Vivo

Cancers ◽

10.3390/cancers13092017 ◽

2021 ◽

Vol 13 (9) ◽

pp. 2017

Author(s):

Lital Sharvit ◽

Rinat Bar-Shalom ◽

Naiel Azzam ◽

Yaniv Yechiel ◽

Solomon Wasser ◽

...

Keyword(s):

Pancreatic Cancer ◽

Cancer Cells ◽

Pancreatic Cancer Cell Line ◽

Cancer Cell Line ◽

Culture Liquid ◽

Human Pancreatic Cancer ◽

P53 Signaling ◽

Pancreatic Cancer Cells

Pancreatic cancer is a highly lethal disease with limited options for effective therapy and the lowest survival rate of all cancer forms. Therefore, a new, effective strategy for cancer treatment is in need. Previously, we found that a culture liquid extract of Cyathus striatus (CS) has a potent antitumor activity. In the present study, we aimed to investigate the effects of Cyathus striatus extract (CSE) on the growth of pancreatic cancer cells, both in vitro and in vivo. The proliferation assay (XTT), cell cycle analysis, Annexin/PI staining and TUNEL assay confirmed the inhibition of cell growth and induction of apoptosis by CSE. A Western blot analysis demonstrated the involvement of both the extrinsic and intrinsic apoptosis pathways. In addition, a RNAseq analysis revealed the involvement of the MAPK and P53 signaling pathways and pointed toward endoplasmic reticulum stress induced apoptosis. The anticancer activity of the CSE was also demonstrated in mice harboring pancreatic cancer cell line-derived tumor xenografts when CSE was given for 5 weeks by weekly IV injections. Our findings suggest that CSE could potentially be useful as a new strategy for treating pancreatic cancer.

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Linc-DYNC2H1-4 promotes EMT and CSC phenotypes by acting as a sponge of miR-145 in pancreatic cancer cells

Cell Death and Disease ◽

10.1038/cddis.2017.311 ◽

2017 ◽

Vol 8 (7) ◽

pp. e2924-e2924 ◽

Cited By ~ 23

Author(s):

Yuran Gao ◽

Zhicheng Zhang ◽

Kai Li ◽

Liying Gong ◽

Qingzhu Yang ◽

...

Keyword(s):

Pancreatic Cancer ◽

Cancer Cells ◽

Ectopic Expression ◽

Pancreatic Cancer Cell Line ◽

Mesenchymal Transition ◽

Gemcitabine Resistance ◽

Pancreatic Cancer Cells ◽

Sense Strand ◽

The Impact

AbstractThe acquisition of epithelial–mesenchymal transition (EMT) and/or existence of a sub-population of cancer stem-like cells (CSC) are associated with malignant behavior and chemoresistance. To identify which factor could promote EMT and CSC formation and uncover the mechanistic role of such factor is important for novel and targeted therapies. In the present study, we found that the long intergenic non-coding RNA linc-DYNC2H1-4 was upregulated in pancreatic cancer cell line BxPC-3-Gem with acquired gemcitabine resistance. Knockdown of linc-DYNC2H1-4 decreased the invasive behavior of BxPC-3-Gem cells while ectopic expression of linc-DYNC2H1-4 promoted the acquisition of EMT and stemness of the parental sensitive cells. Linc-DYNC2H1-4 upregulated ZEB1, the EMT key player, which led to upregulation and downregulation of its targets vimentin and E-cadherin respectively, as well as enhanced the expressions of CSC makers Lin28, Nanog, Sox2 and Oct4. Linc-DYNC2H1-4 is mainly located in the cytosol. Mechanically, it could sponge miR-145 that targetsZEB1,Lin28,Nanog,Sox2,Oct4to restore these EMT and CSC-associated genes expressions. We proved thatMMP3, the nearby gene of linc-DYNC2H1-4 in the sense strand, was also a target of miR-145. Downregulation ofMMP3by miR-145 was reverted by linc-DYNC2H1-4, indicating that competing with miR-145 is one of the mechanisms for linc-DYNC2H1-4 to regulateMMP3. In summary, our results explore the important role of linc-DYNC2H1-4 in the acquisition of EMT and CSC, and the impact it has on gemcitabine resistance in pancreatic cancer cells.

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Possible role of autophagy inhibition in hypoxia-induced chemoresistance of pancreatic cancer cells.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.4_suppl.224 ◽

2012 ◽

Vol 30 (4_suppl) ◽

pp. 224-224 ◽

Cited By ~ 4

Author(s):

Yoon Ho Ko ◽

Young-Seok Cho ◽

Hye Sung Won ◽

Eun Kyoung Jeon ◽

Young Seon Hong

Keyword(s):

Pancreatic Cancer ◽

Cancer Cells ◽

Western Blotting ◽

Pancreatic Cancer Cell Line ◽

Cancer Cell Line ◽

Hypoxic Condition ◽

Human Pancreatic Cancer ◽

Pancreatic Cancer Cells ◽

Autophagy Inhibition

224 Background: Autophagy is a catabolic process and provides metabolic support for the cell by degradation of intracellular macromolecules. Various types of stress, including hypoxia, activate autophagy. Recent studies have suggested that hypoxia has been shown to associate with resistance to chemotherapy and radiation therapy and hence poor prognosis in pancreatic cancer. This study investigated the role of autophagy in the treatment of pancreatic cancer with gemcitabine under hypoxic condition. Methods: To evaluate the role of autophagy inhibition in hypoxia-induced chemoresistance, BxPC-3 human pancreatic cancer cell line was used under normoxic and hypoxic conditions.We evaluated the extent of LC3-II, as an autophagosome marker, induced by gemcitabine, by western blotting to measure the hypoxia- or chemotherapy- induced autophagy. We then examined the effects of gemcitabine on induction of apoptosis under normoxic and hypoxic conditions. Next, to determine the effect of 3-MA, a known inhibitor of autophagy, on overcoming hypoxia-induced chemoresistance, the MTS assay and flow cytometry were performed. Results: Compared with normoxia, gemcitabine-induced cell death under hypoxia was significantly decreased, as a result of the reduced apoptosis. Western blotting analysis demonstrated that LC3-II was increased under hypoxia, compared with normoxia.However, we found that 3-MA can enhance the growth inhibition and apoptotic effect of gemcitabine, even under hypoxia. These findings mean that autophagy mediates the chemoresistance under hypoxia. Conclusions: Activated autophagy plays a role in hypoxia-induced chemoresistance of pancreatic cancer cells. These findings may have important implications for future therapeutic strategies using gemcitabine against pancreatic cancer.

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Propofol Mediates Pancreatic Cancer Cell Activity Through the Repression of ADAM8 via SP1

10.21203/rs.3.rs-150729/v1 ◽

2021 ◽

Author(s):

Yutong Gao ◽

Yu Zhou ◽

Chunlin Wang ◽

Klarke Sample ◽

Xiangdi Yu ◽

...

Keyword(s):

Pancreatic Cancer ◽

Cancer Cells ◽

Cancer Cell ◽

Pancreatic Cancer Cell ◽

Cell Activity ◽

Pancreatic Cancer Cell Line ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Physical Interaction ◽

Pancreatic Cancer Cells

Abstract Background Propofol is a commonly used anesthetic with controversial effects on cancer cells. A growing number of studies have demonstrated that low concentrations of propofol are associated with tumor suppression and when used as an intravenous anesthesia improved recurrence-free survival rates for many cancers, but deeper insights into its underlying mechanism are needed. Methods The study detailed herein focuses upon the effect of propofol on pancreatic cancer cells and the mechanism by which propofol reduces ADAM8 expression. The ability of propofol to impact the proliferation, migration and cell cycle of a pancreatic cancer cell line was assessed in vitro. This was mechanistically explored following the identification of SP1 binding sites within ADAM8, which enabled the regulatory effects of SP1 on ADAM8 following propofol treatment to be further explored. Results This study was able to show that propofol significantly inhibited the proliferation, migration and invasion of pancreatic cancer cells and decreased the percentage of cells in S-phase. Propofol treatment was also shown to repress ADAM8 and SP1 expression, but was unable to affect ADAM8 expression following knockdown of SP1. Moreover, a direct physical interaction between SP1 and ADAM8 was verified using Co-immunoprecipitation and dual-luciferase reporter assays. Conclusion These results suggest that propofol represses pathological biological behaviors associated with pancreatic cancer cells through the suppression of SP1, which in turn results in lower ADAM8 mRNA expression and protein levels.

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