BackgroundCancer stem cells (CSCs) are regarded as the cause of tumor formation and recurrence. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs.MethodsHuman hepatoma cell lines were plated in stem cell conditioned culture system allowed for sphere forming. To evaluate the stemness characteristics of spheres, the self-renewal, proliferation, chemoresistance, tumorigenicity of the PLC/PRF/5 sphere-forming cells, and the expression levels of stem cell related proteins in the PLC/PRF/5 sphere-forming cells were assessed, comparing with the parental cells. The stem cell RT-PCR array was performed to further explore the biological properties of liver CSCs.ResultsThe PLC/PRF/5, MHCC97H and HepG2 cells could form clonal nonadherent 3-D spheres and be serially passaged. The PLC/PRF/5 sphere-forming cells possessed a key criteria that define CSCs: persistent self-renewal, extensive proliferation, drug resistance, overexpression of liver CSCs related proteins (Oct3/4, OV6, EpCAM, CD133 and CD44). Even 500 sphere-forming cells were able to form tumors in NOD/SCID mice, and the tumor initiating capability was not decreased when spheres were passaged. Besides, downstream proteins DTX1 and Ep300 of the CSL (CBF1 in humans, Suppressor of hairless in Drosophila and LAG1 in C. elegans) -independent Notch signaling pathway were highly expressed in the spheres, and a gamma-secretase inhibitor MRK003 could significantly inhibit the sphere formation ability.ConclusionsNonadherent tumor spheres from hepatoma cell lines cultured in stem cell conditioned medium possess liver CSC properties, and the CSL-independent Notch signaling pathway may play a role in liver CSCs.

Purpose: To establish a sensitive and specific isolation and enumeration system for circulating tumor cells (CTC) in patients with hepatocellular carcinoma (HCC).Experimental Design: HCC cells were bound by biotinylated asialofetuin, a ligand of asialoglycoprotein receptor, and subsequently magnetically labeled by antibiotin antibody-coated magnetic beads, followed by magnetic separation. Isolated HCC cells were identified by immunofluorescence staining using Hep Par 1 antibody. The system was used to detect CTCs in 5 mL blood. Blood samples spiked with Hep3B cells (ranging from 10 to 810 cells) were used to determine recovery and sensitivity. Prevalence of CTCs was examined in samples from HCC patients, healthy volunteers, and patients with benign liver diseases or non-HCC cancers. CTC samples were also analyzed by FISH.Results: The average recovery was 61% or more at each spiking level. No healthy, benign liver disease or non-HCC cancer subjects had CTCs detected. CTCs were identified in 69 of 85 (81%) HCC patients, with an average of 19 AE 24 CTCs per 5 mL. Both the positivity rate and the number of CTCs were significantly correlated with tumor size, portal vein tumor thrombus, differentiation status, and the disease extent as classified by the TNM (tumor-node-metastasis) classification and the Milan criteria. HER-2 gene amplification and TP53 gene deletion were detected in CTCs.Conclusion: Our system provides a new tool allowing for highly sensitive and specific detection and genetic analysis of CTCs in HCC patients. It is likely clinically useful in diagnosis and monitoring of HCC and may have a role in clinical decision making.

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BackgroundOCT4 and BIRC5 are preferentially expressed in human cancer cells and mediate cancer cell survival and tumor maintenance. However, the molecular mechanism that regulates OCT4 and BIRC5 expression is not well characterized.MethodsBy manipulating OCT4 and BIRC5 expression in hepatocellular carcinoma (HCC) cell lines, the regulatory mechanism of OCT4 on BIRC5 and CCND1 were investigated.ResultsIncreasing or decreasing OCT4 expression could enhance or suppress BIRC5 expression, respectively, by regulating the activity of BIRC5 promoter. Because there is no binding site for OCT4 within BIRC5 promoter, the effect of OCT4 on BIRC5 promoter is indirect. An octamer motif for OCT4 in the CCND1 promoter has directly and partly participated in the regulation of CCND1 promoter activity, suggesting that OCT4 also could upregulated the expression of CCND1. Co-suppression of OCT4 and BIRC5 induced cancer cell apoptosis and cell cycle arrest, thereby efficiently inhibiting the proliferative activity of cancer cells and suppressing the growth of HCC xenogrfts in nude mice.ConclusionOCT4 can upregulate BIRC5 and CCND1 expression by increasing their promoter activity. These factors collusively promotes HCC cell proliferation, and co-suppression of OCT4 and BIRC5 is potentially beneficial for HCC treatment.

This study shows that ZEB1 high expression was correlated with HCC malignant progression and subsequent poor patient survival by induction of EMT changes.

Chilling tolerance was increased in seed germination and root growth of wheat seedlings grown in media containing 20 µg/mL cerebroside C (CC), isolated from the endophytic Phyllosticta sp. TG78. Seeds treated with 20 µg/mL CC at 4°C expressed the higher germination rate (77.78%), potential (23.46%), index (3.44) and the shorter germination time (6.19 d); root growth was also significantly improved by 13.76% in length, 13.44% in fresh weight and 6.88% in dry mass compared to controls. During the cultivation process at 4°C for three days and the followed 24 h at 25°C, lipid peroxidation, expressed by malondialdehyde (MDA) content and relative membrane permeability (RMP) was significantly reduced in CC-treated roots; activities of lipoxygenase (LOX), phospholipid C (PLC) and phospholipid D (PLD) were inhibited by 13.62–62.26%, 13.54–63.93% and 13.90–61.17%, respectively; unsaturation degree of fatty acids was enhanced through detecting the contents of CC-induced linoleic acid, linolenic acid, palmitic acid and stearic acid using GC-MS; capacities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were individually increased by 7.69–46.06%, 3.37–37.96%, and −7.00–178.07%. These results suggest that increased chilling tolerance may be due, in part, to the reduction of lipid peroxidation and alternation of lipid composition of roots in the presence of CC.

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X-ray phase contrast imaging (PCI) based on synchrotron radiation was introduced for the first time as an in situ imaging way to investigate sea salt phase change, i.e. deliquescence and crystallization in atmospheric environment. A performance on the deliquescence of pure NaCl, which is the dominant component in sea salt, demonstrated that this technique can directly observe the change of core particle and differentiate the outer water layer clearly in solid-aqueous system of~100 mm scale. The imaging results showed that sea salt particle deliquesced on a large scale of relative humidity (RH) between 34 and 97% RH as a solid-liquid drop, while no clear deliquescence RH was observed during the process. According to the drop size growth curve, sea salt deliquescence can be divided into three steps, namely water accumulating step (34-75% RH), bulk melting step (75-86% RH) and delay dissolving step (>86% RH), which are most probably dominated by grouped components as MgCl 2 / CaCl 2 /MgSO 4 , NaCl and Na 2 SO 4 /KCl/K 2 SO 4 , respectively. Instead at a sole RH, the crystallization of sea salt solution occurred at a range of 46-58% RH, which well agreed with the theory proposed by Ge et al. The aqueous-solid condition provided by sea salt deliquescence and crystallization may greatly enhance the heterogeneous chemical reactions in atmosphere.