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Human bestrophin-1 (hBest1), which is genetically linked to several kinds of retinopathy and macular degeneration in both humans and dogs, is the founding member of a family of [C1.sup.-1] ion channels that are activated by intracellular [Ca.sup.2+]. At present, the structures and mechanisms responsible for [Ca.sup.2+] sensing remain unknown. Here, we have used a combination of molecular modeling, density functional--binding energy calculations, mutagenesis, and patch clamp to identify the regions of hBest1 involved in [Ca.sup.2+] sensing. We identified a cluster of a five contiguous acidic amino acids in the C terminus immediately after the last transmembrane domain, followed by an EF hand and another regulatory domain that are essential tot [Ca.sup.2+] sensing by hBest1. The cluster of five amino acids (293-308) is crucial for normal channel gating by [Ca.sup.2+] because all but two of the 35 mutations we made in this region rendered the channel incapable of being activated by [Ca.sup.2+]. Using homology, models built on the crystal structure of calmodulin (CAM), an EF hand (EF1) was identified in hBest1. EF1 was predicted to bind [Ca.sup.2+] with a slightly higher affinity than the third EF hand of CaM and lower affinity than the second EF hand of troponin C. As predicted by the model, the D312G mutation in the putative [Ca.sup.2+]-binding loop (312-323) reduced the apparent [Ca.sup.2+] affinity by 20-fold. In addition, the D312G and D323N mutations abolished [Ca.sup.2+]-dependent rundown of the current. Furthermore, analysis of truncation mutants of hBest1 identified a domain adjacent to EF1 that is rich in acidic amino acids (350-390) that is required for [Ca.sup.2+] activation and plays a role in current rundown. These experiments identity a region of hBest1 (312-323) that is involved in the gating of hBest1 by [Ca.sup.2+] and suggest a model in which [Ca.sup.2+] binding to EF1 activates the channel in a process that requires the acidic domain (293-308) and another regulatory domain (350-390). Many of the ~100 disease-causing mutations in hBest1 are located in this region that we have implicated in [Ca.sup.2+] sensing, suggesting that these mutations disrupt hBestl channel gating by [Ca.sup.2+].

Background and Purpose: Ca2+‐activated Cl− channels (Ano1 channels) contribute to the pathogenesis of colorectal cancer. Honokiol is known to inhibit cell proliferation and tumour growth in colorectal cancer. However, the molecular target of honokiol remains unclear. This study aimed to investigate whether honokiol inhibited cell proliferation of colorectal cancer by targeting Ano1 channels. Experimental Approach Patch‐clamp techniques were performed to study the effect of honokiol on Ca2+‐activated Cl− currents in HEK293 cells overexpressing Ano1‐ or Ano2‐containing plasmids or in human colorectal carcinoma SW620 cells. Site‐directed mutagenesis was used to identify the critical residues for honokiol‐induced Ano1 inhibition. Proliferation of SW620 cells or human intestinal epithelial NCM460 cells by CCK‐8 assays. Key Results: Honokiol blocked Ano1 currents in Ano1‐overexpressing HEK293 cells and SW620 cells. Honokiol more potently inhibited Ano1 currents than Ano2 currents. Three amino acids (R429, K430 and N435) were critical for honokiol‐induced Ano1 inhibition. The R429A/K430L/N435G mutation reduced the sensitivity of Ano1 to honokiol. Honokiol inhibited SW620 cell proliferation, and this effect was reduced by Ano1‐shRNAs. Furthermore, Ano1 overexpression promoted proliferation in NCM460 cells with low Ano1 endogenous expression and resulted in an increased sensitivity to honokiol. Overexpression of the R429A/K430L/N435G mutation reduced WT Ano1‐induced increase in the sensitivity of NCM460 cells to honokiol. Conclusion and Implications: We identified a new anticancer mechanism of honokiol, through the inhibition of cell proliferation, by targeting Ano1 Ca2+‐activated Cl− channels. [ABSTRACT FROM AUTHOR]

Double strand breaks induced by genotoxic agents, if inappropriately repaired, will cause cell death or induce cancer. Poly(ADP-ribose) polymerase-3 (PARP-3) serves a role in double strand break repair, and may be involved in tumorigenesis. To the best of our knowledge, the role of PARP-3 in breast cancer has not yet been examined. In the present study, the expression of PARP-3 was investigated in 493 breast cancer samples and 54 tumor-adjacent control samples using tissue-microarray-based immunohistochemistry. PARP-3 expression was higher in breast cancer samples compared with control samples. PARP-3 overexpression was significantly associated with histological grade II–III (P=0.012). In addition, PARP-3 overexpression was significantly associated with shorter disease-free survival (DFS; P=0.027) time and exhibited a tendency toward shorter overall survival (OS; P=0.183) time in patients with breast cancer compared with patients with lower PARP-3 expression, particularly in BRCA1-positive patients (P=0.004 for disease-free survival and P=0.095 for OS). Multivariate Cox regression analysis indicated that PARP-3 was an independent prognostic factor in patients with breast cancer. Furthermore, it was revealed that PARP-3 overexpression was associated with shorter survival time in patients with cyclophosphamide/doxorubicin or epirubicin/5-fluorouracil (CAF/CEF) chemotherapy compared with low PARP-3 expression, but not in patients with CAF/CEF + docetaxel chemotherapy. The present study suggested that PARP-3 may be used as a biomarker for predicting the clinical outcome of patients receiving chemotherapy, and targeting PARP-3 may be a potential therapeutic strategy for the treatment of breast cancer. [ABSTRACT FROM AUTHOR]

Enhancer of zeste homologue 2 (EZH2), a catalytic subunit of polycomb repressive complex 2, is overexpressed in a number of different tumors including breast cancer, and serves important roles in cell cycle regulation, proliferation, apoptosis, tumorigenesis and drug resistance. However, it remains unclear whether EZH2 contributes to tamoxifen resistance in breast cancer. In the present study, the role of EZH2 in tamoxifen resistance in MCF-7 cells was investigated. EZH2 was overexpressed in MCF-7 tamoxifen-resistant (MCF-7 TamR) cells. EZH2 overexpression decreased the sensitivity of MCF-7 cells to tamoxifen, and EZH2 knockdown improved the sensitivity of MCF-7 TamR cells to tamoxifen. Furthermore, EZH2 knockdown induced cell cycle arrest in MCF-7 TamR cells, accompanied by a decrease in cyclin D1 expression and an increase in p16 expression. EZH2 knockdown reduced p16 gene methylation in MCF-7 TamR cells. These findings suggested that EZH2 overexpression may contribute to tamoxifen resistance in breast cancer, and EZH2 inhibition may reverse tamoxifen resistance in breast cancer by regulating the cell cycle via the demethylation of the p16 gene. Thus, EZH2 inhibitors may be effective for treating tamoxifen resistance in breast cancer. [ABSTRACT FROM AUTHOR]

TMEM16A, also known as anoctamin 1, is a recently identified Ca2+-activated chloride channel and the first member of a 10-member TMEM16 family. TMEM16A dysfunction is implicated in many diseases such as cancer, hypertension, and cystic fibrosis. TMEM16A channels are well known to be dually regulated by voltage and Ca2+. In addition, recent studies have revealed that TMEM16A channels are regulated by many molecules such as calmodulin, protons, cholesterol, and phosphoinositides, and a diverse range of stimuli such as thermal and mechanical stimuli. A better understanding of the regulatory mechanisms of TMEM16A is important to understand its physiological and pathological role. Recently, the crystal structure of a TMEM16 family member from the fungus Nectria haematococcaten (nhTMEM16) is discovered, and provides valuable information for studying the structure and function of TMEM16A. In this review, we discuss the structure and function of TMEM16A channels based on the crystal structure of nhTMEM16A and focus on the regulatory mechanisms of TMEM16A channels. J. Cell. Physiol. 232: 707-716, 2017. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]