Metastasis leads to poor prognosis in colorectal cancer patients, and there is a growing need for new therapeutic targets. TMEM16A (ANO1, DOG1 or TAOS2) has recently been identified as a calcium-activated chloride channel (CaCC) and is reported to be overexpressed in several malignancies; however, its expression and function in colorectal cancer (CRC) remains unclear. In this study, we found expression of TMEM16A mRNA and protein in high-metastatic-potential SW620, HCT116 and LS174T cells, but not in primary HCT8 and SW480 cells, using RT-PCR, western blotting and immunofluorescence labeling. Patch-clamp recordings detected CaCC currents regulated by intracellular Ca2+ and voltage in SW620 cells. Knockdown of TMEM16A by short hairpin RNAs (shRNA) resulted in the suppression of growth, migration and invasion of SW620 cells as detected by MTT, wound-healing and transwell assays. Mechanistically, TMEM16A depletion was accompanied by the dysregulation of phospho-MEK, phospho-ERK1/2 and cyclin D1 expression. Flow cytometry analysis showed that SW620 cells were inhibited from the G1 to S phase of the cell cycle in the TMEM16A shRNA group compared with the control group. In conclusion, our results indicate that TMEM16A CaCC is involved in growth, migration and invasion of metastatic CRC cells and provide evidence for TMEM16A as a potential drug target for treating metastatic colorectal carcinoma.

Elevated blood cholesterol is an important risk factor associated with atherosclerosis and coronary heart disease. The search for mediators that fine-tune cholesterol homeostasis has revealed lactic acid bacteria (LAB) to be potentially beneficial. The aim of the present study was to identify and characterize probiotic strains with bile salt hydrolase activity from kefir grains and evaluate their potential use as cholesterol-reducing probiotics in rats. Two isolates, Lp09 and Lp45, obtained from kefir grains were identified as Lactobacillus plantarum via molecular typing methods. Lactobacillus plantarum Lp09 and Lp45 exhibited excellent tolerance to low pH levels and high bile salt concentrations and showed potential bile salt hydrolase activity, bile salt deconjugation activity, and cholesterol coprecipitation ability. Additionally, the potential effect of Lb. plantarum Lp09 and Lp45 on plasma cholesterol levels was evaluated in Sprague-Dawley rats. Rats in 3 treatment groups were fed different experimental diets: a high-cholesterol diet, a high-cholesterol diet plus Lb. plantarum Lp09, or a high-cholesterol diet plus Lb. plantarum Lp45 for 4 wk. Total cholesterol, triglyceride, and low-density lipoprotein cholesterol levels in serum as well as cholesterol and triglyceride levels in liver were significantly decreased in the LAB-treated rats compared with rats fed a high-cholesterol diet without LAB supplementation. Also, both fecal cholesterol and bile acid levels were significantly increased after LAB administration. No significant changes were detected in high-density lipoprotein cholesterol levels. These results suggest that the Lb. plantarum Lp09 and Lp45 strains present the potential to be explored as probiotic agents for the management of hypercholesterolemia.

Lactobacillus plantarum Lp27 was isolated from Tibetan kefir grains. The Lp27 isolate survived a 3-h incubation at pH 2.0 and grew normally in 0.3% oxgall. In addition, the Lp27 isolate exhibited an adhesion ratio of 9.5 ± 2.5% with Caco-2 cells. Antibiotic susceptibility tests indicated that the Lp27 isolate was sensitive to gentamicin, tetracycline, erythromycin, and chloramphenicol, and was resistant to vancomycin with a minimum inhibitory value of 23µg/mL. The Lp27 isolate inhibited cholesterol absorption through downregulation of Niemann-Pick C1-like 1 (NPC1L1) expression in Caco-2 cells. The Lp27 isolate was fed to hypercholesterolemic rats at a dose of 10(9) cfu/d for 4wk. The Lp27 feeding significantly lowered serum total cholesterol, low-density lipoprotein cholesterol, and triglycerides concentrations, but no change was observed in the serum high-density lipoprotein cholesterol concentrations. In addition, liver total cholesterol and triglycerides were decreased in the Lp27-fed group. The expression of NPC1L1 in the duodenum and jejunum was significantly decreased following Lp27 feeding. These results indicate that Lp27 might be an effective cholesterol-lowering probiotic and a possible mechanism for the cholesterol-reducing effects of probiotics.

Candida albicans infections present a heavy burden upon public health, with only a few drugs available, while biofilms formed by C. albicans worsen this situation. Dioscin has antitumor, anti-inflammatory, and hepatoprotective effects, and this study was conducted to evaluate the effects of dioscin on the biofilm formation and development, as well as other virulence factors of C. albicans such as morphological transition, adhesion, and extracellular secreted phospholipase. Our results showed dioscin inhibits these virulence factors and has low cytotoxicity against mammalian cells. Considering protective effects of dioscin against damage on liver and kidney, dioscin may be used as a potential candidate for antifungal development.

Abstract.Human osteosarcoma is an aggressive tumor which frequently resists chemotherapy; therefore, the search for new agents for its treatment is of great importance. Isoalantolactone, isolated from Inula spp., has been reported to inhibit the growth of several types of cancer cells. However, no prior research has been conducted to demonstrate the antiproliferative potential of isoalantolactone on osteosarcoma. The present study is the first to investigate the effects of isoalantolactone on cell viability in human osteosarcoma U2OS, MG-63 and Saos-2 cells, and its mechanism of action in U2OS cells. Our results demonstrated that isoalantolactone triggered S and mainly G2/M cell cycle phase arrest, accompanied by the downregulation of the expression of cyclin B1 at the protein and mRNA levels. Moreover, isoalantolactone induced apoptosis that was associated with reactive oxygen species (ROS) generation and the dissipation of mitochondrial membrane potential (MMP). Furthermore, our results indicated that this compound upregulated DR5, FADD and cleaved caspase-8, increased the interation between DR5 and FADD, and inhibited the expression of nuclear NF-κBp65. We also found that isoalantolactone-induced apoptosis was associated with the downregulation of Bcl-2 and upregulation of Bax, which finally led to the activation of caspase-3 and its downstream substrate, PARP, in osteosarcoma U2OS cells. Isoalantolactone-induced apoptosis was markedly abrogated when the cells were pretreated with N-acetylcysteine (NAC), a specific ROS inhibitor, suggesting that the apoptosis-inducing effect of isoalantolactone in osteosarcoma cells was mediated by reactive oxygen species. Taken together, our data demonstrated that isoalantolactone induces ROS-dependent apoptosis in U2OS cells via a novel mechanism involving inhibition of NF-κBp65 and provide the rationale for further in vivo and preclinical investigation of isoalantolactone against osteosarcoma.

KRAS, NRAS and BRAF are kinases involved in the RAS-RAF-MAPK signaling pathway and also potential tumor-driven genes. Patients with KRAS/NRAS/BRAF mutations are resistant to anti-EGFR monoclonal antibody therapy. The main purpose of this study is to investigate the mutation status and distribution of KRAS/NRAS/BRAF in Chinese colorectal and gastric cancers, and to explore the histopathological changes and related immunohistochemical marker changes caused by these mutations. The mutation status of KRAS (exons 2, codon 12/13), NRAS (exons 2/3/4, codon 12/13/59/61/117/146) and BRAF (exons 15, codon 600) were detected by amplification refractory mutation system polymerase chain reaction (ARMS-PCR) in 86 colon cancer, 140 rectal cancer and 34 gastric cancer tissues. Then, the frequencies and distribution of KRAS/NRAS/BRAF mutations were described in detail. Furthermore, the relationship between KRAS/NRAS/BRAF mutations and the features of histopathological and related immunohistochemical markers were analyzed. The results showed that KRAS/NRAS/BRAF mutation rates in colon cancer were 44.2, 1.2, and 3.5%; in rectal cancer were 37.1, 4.3, and 0.7%; in gastric cancer were none, none and 2.9%. The mutation rate of KRAS in female (48.8%) is significantly higher than that of male (27.8%), and the mutation rate increased with the higher degree of differentiation. Additionally, the mutation rate of BRAF detected by ARMS-PCR (1.77%) was significantly lower than that by immunohistochemistry (4.11%). It also showed that the KRAS/NRAS/BRAF mutation status had a certain relationship with the expression of some immunohistochemical markers. This study provides more data support for clinical research on KRAS/NRAS/BRAF mutation in CRCs or gastric cancers.

TMEM16A, a calcium-activated chloride channel (CaCC), is highly amplified and expressed in human cancers and is involved in the growth and metastasis of some malignancies. Inhibition of TMEM16A represents a novel pharmaceutical approach for the treatment of cancers and metastases. The purpose of this study is to identify a new TMEM16A inhibitor, investigate the effects of this inhibitor on the proliferation and metastasis of TMEM16A-amplified SW620 cells, and to elucidate the underlying molecular mechanism in vitro. We identified a novel small-molecule TMEM16A inhibitor dehydroandrographolide (DP). By using patch clamp electrophysiology, we showed that DP inhibited TMEM16A chloride currents in Fisher rat thyroid (FRT) cells that were transfected stably with human TMEM16A and in TMEM16A-overexpressed SW620 cells but did not alter cystic fibrosis transmembrane conductance regulator (CFTR) chloride currents. Further functional studies showed that DP suppressed the proliferation of SW620 cells in a dose- and time-dependent manner using MTT assays. Moreover, DP significantly inhibited migration and invasion of SW620 cells as detected by wound-healing and transwell assays. Further mechanistic study demonstrated that knockdown of human TMEM16A decreased the inhibitory effect of DP on the proliferation of SW620 cells and that TMEM16A-dependent cells (SW620 and HCT116) were more sensitive to DP than TMEM16A-independent cells (SW480 and HCT8). In addition, we found that treatment of SW620 cells with DP led to a decrease in TMEM16A protein levels but had no effect on TMEM16A mRNA levels. The current work reveals that DP, a novel TMEM16A inhibitor, exerts its anticancer activity on SW620 cells partly through a TMEM16A-dependent mechanism, which may introduce a new targeting approach for an antitumour therapy in TMEM16A-amplified cancers.

This study aims to understand what older Chinese people with chronic illness and their family caregivers perceive to be good care, and to compare perspectives of those living in rural and urban areas. We conducted semistructured interviews with 24 care recipients and 23 caregivers in Shandong, China. Two major themes were identified: (a) filial piety as the standard, and (b) modifying cultural ideals to meet reality. There was overall consistency in perceptions of study participants. Variations between rural and urban elders' perceptions appear to reflect differences in socioeconomic development and institutional structures.