Isolation of Small Extracellular Vesicles From Human Serum Using a Combination of Ultracentrifugation With Polymer-Based Precipitation

Methods for reproducibly isolating and enriching small extracellular vesicles (EVs) from blood are essential for clinical utilization of small EVs in cancer patients. We combined ultracentrifugation (UC) with polymer-based precipitation (ExoQuick [EQ] or Total Exosome Isolation [TEI] kit) to isolate small EVs (diameter, 30–150 nm) from the serum of breast cancer patients. We compared the performance of four cycles of UC (UC4x) with that of two cycles of UC followed by enrichment using the EQ (UC2x→EQ) or TEI (UC2x→TEI) kits. The mean concentration of small EVs isolated from 1 mL of serum using UC2x→EQ (139.0±29.1 µg) and UC2x→TEI (140.4±5.0 µg) did not differ from that obtained using UC4x (141.8±26.9 µg). The mean number of EV particles obtained using UC4x was 29.2±9.9×109 per mL of serum, whereas UC2x→EQ and UC2x→TEI yielded higher numbers of EVs (50.7±17.0×10⁹ and 59.3±20.6×10⁹, respectively). Concentrations of EV microRNAs, including miR-21 and miR-155, did not differ between the three methods. In conclusion, performing UC prior to the use of polymer-based precipitation kits could be feasible for isolating small EVs from human serum in large sample-based translational researches.

Maspin Suppresses Survival of Lung Cancer Cells through Modulation of Akt Pathway / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Maspin is a tumor suppressor protein that has been reported to stimulate the cell death of cancer and inhibit the metastasis of cancer. The present study aimed to explore the survival pathway by which maspin modulates the resistance of human lung cancer cells to chemotherapeutic drugs, and the consequences of maspin gene therapy in an animal model. MATERIALS AND METHODS: NCI-H157 and A549 cells were transfected with either a mock vector (pCMVTaq4C), maspin (pCMV-maspin), siControl or siMaspin. RT-PCR and Western blot analysis were performed to study the expressions of survival proteins in lung cancer. cDNA microarray analysis was carried out to compare the maspin-modulated gene expression between the xenograft tumors derived from the lung cancer cells that were stably transfected with pCMVTaq4C or pCMV-maspin. Maspin gene therapy was performed by intra-tumoral injections of pCMVTaq4C or pCMV-maspin into the pre-established subcutaneous tumors in nude mice. RESULTS: Maspin significantly decreased the survival to doxorubicin and etoposide, whereas did not affect the survival to cisplatin in the NCI-H157 cells. Interestingly, transfection with a maspin plasmid resulted in a significant reduction of the phosphorylation of Akt in the NCI-H157 cells, whereas knockdown of maspin increased the phosphorylation of Akt in the A549 cells. Microarray analysis of the xenograft tumors revealed a specific gene expression profile, demonstrating that maspin is associated with the differential expressions of PTEN and IGF2R. Direct transfer of pCMV-maspin into the tumor significantly retarded the tumor growth in the animal experiments (p=0.0048). CONCLUSION: Lung cancer cells lacking maspin could be resistant to chemotherapeutic drugs such as doxorubicin or etoposide, at least in part by maintaining Akt phosphorylation.

Enhancement of Plasmacytoma Cell Growth by Ascorbic Acid is Mediated Via Glucose 6-phosphate Dehydrogenase / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: We investigated the mechanism by which some types of cancer cells grow faster in the presence of ascorbic acid supplementation. MATERIALS AND METHODS: Adj.PC-5, a mouse plasmacytoma cell, is known to show ascorbic acid-dependent growth and was chosen as a test system. The growth of cancer cells was measured by the colony number on soft agar or the cellular proliferation in suspension culture. The ascorbate level was measured by a high performance liquid chromatography system with an electrochemical detector. Glucose 6-phosphate dehydrogenase was analyzed both on the specific enzyme activity level and on the transcription level by performing Northern blot analysis. RESULTS: Ascorbyl 2-phosphate among the ascorbate derivatives was the most efficient in stimulating cell growth. The intracellular and extracellular ascorbate concentrations following treatment with either ascorbate or ascorbyl 2-phosphate suggest that the superiority of ascorbyl 2-phosphate for stimulating cell growth may be due to its slow conversion to ascorbate in the culture medium. The steady transformation to ascorbate ensures sustained levels of ascorbate in the culture medium and thereby maximizes the growth stimulatory effect of ascorbate. Ascorbyl 2-phosphate markedly enhanced, in a concentration-and time-dependent manner, mRNA synthesis as well as the enzymatic activity of glucose 6- phosphate dehydrogenase, which is known to be a rate- limiting enzyme in cell growth. On the other hand, simultaneous addition of dehydroisoandrosterone, a well- known inhibitor of glucose 6-phosphate dehydrogenase, to the culture medium abrogated the growth stimulation by ascorbyl 2-phosphate, and it also reduced the glucose 6-phosphate dehydrogenase activity proportionately. CONCLUSIONS: The results from this study suggest that enhanced glucose 6-phosphate dehydrogenase activity may at least in part explain the stimulation of cell growth by ascorbate or ascorbyl 2-phosphate.

Characterization of RhoA-mediated Chemoresistance in Gastric Cancer Cells / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: RhoA is a critical transducer of extracellular signals, which leads to organization of actin cytoskeleton, motility, adhesion and gene regulation. The present study aimed to explore whether RhoA influences the susceptibility of gastric cancer cells to chemotherapeutic drugs. MATERIALS AND METHODS: SNU638 cells were transfected with a mock vector (pcDNA3.1), RhoA (pcDNA/RhoA), or constitutively active RhoA (pcDNA/caRhoA). MTT assay and Western blot analysis were performed to study the growth response to several chemotherapeutic drugs in the gastric cancer cell line, SNU638, with different RhoA levels. RESULTS: RhoA significantly enhanced the resistance to lovastatin, 5-FU, taxol and vincristine, but did not affect the sensitivity to cisplatin or etoposide in SNU638. In the Western blot analysis, RhoA decreased the PARP cleavage, which was accompanied by a concurrent reduction in cell death. The gene expression profile after a cDNA microarray analysis demonstrated that RhoA was associated with the differential expression of 19 genes, including those involved in anti-oxidant defense, glucose metabolism, anti-apoptosis and protein turnover. CONCLUSION: Gastric cancer cells with a high expression of RhoA could be resistant to chemotherapeutic drugs, such as taxol or vincristine, implying that treatment strategies aimed at inactivation of RhoA might be promising for improving the efficacy of these chemotherapeutic drugs.

Effects of Lovastatin in Combination with 5-FU on Stomach Cancer Cells / 대한암학회지

No abstract available