Bitter taste receptors: Novel insights into the biochemistry and pharmacology

Bitter taste receptors (T2Rs) belong to the super family of G protein-coupled receptors (GPCRs). There are 25 T2Rs expressed in humans, and these interact with a large and diverse group of bitter ligands. T2Rs are expressed in many extra-oral tissues and can perform diverse physiological roles. Structure-function studies led to the identification of similarities and dissimilarities between T2Rs and Class A GPCRs including amino acid conservation and novel motifs. However, the efficacy of most of the T2R ligands is not yet elucidated and the biochemical pharmacology of T2Rs is poorly understood. Recent studies on T2Rs characterized novel ligands including blockers for these receptors that include inverse agonist and antagonists. In this review we discuss the techniques used for elucidating bitter blockers, concept of ligand bias, generic amino acid numbering, the role of cholesterol, and conserved water molecules in the biochemistry and pharmacology of T2Rs.     

Bitter taste receptor T2R1 is activated by dipeptides and tripeptides

Bitter taste signaling in humans is mediated by a group of 25 bitter receptors (T2Rs) that belong to the G-protein coupled receptor (GPCR) family. Previously, several bitter peptides were isolated and characterized from bitter tasting food protein derived extracts, such as pea protein and soya bean extracts. However, the molecular targets or receptors in humans for these bitter peptides were poorly characterized and least understood. In this study, we tested the ability of the bitter tasting tri- and di-peptides to activate the human bitter receptor, T2R1. In addition, we tested the ability of peptide inhibitors of the blood pressure regulatory protein, angiotensin converting enzyme (ACE) to activate T2R1. Using a heterologous expression system, T2R1 gene was transiently expressed in C6-glioma cells and changes in intracellular calcium was measured following addition of the peptides. We found that the bitter tasting tri-peptides are more potent in activating T2R1 than the di-peptides tested. Among the peptides examined, the bitter tri-peptide Phe-Phe-Phe (FFF), is the most potent in activating T2R1 with an EC₅₀ value in the micromolar range. Furthermore, to elucidate the potential ligand binding pocket of T2R1 we used homology molecular modeling. The molecular models showed that the bitter peptides bind within the same binding pocket on the receptor. The ligand binding pocket in T2R1 is present on the extracellular surface of the receptor, and is formed by the transmembrane helices 1, 2, 3 and 7 and with extracellular loops 1 and 2 forming a cap like structure on the binding pocket.     

Modulation of tumorigenesis and oestrogen receptor‐α expression by cell culture conditions in a stem cell‐derived breast epithelial cell line

Background information. The common phenotypes of cancer and stem cells suggest that cancers arise from stem cells. Oestrogen is one of the few most important determinants of breast cancer, as shown by several lines of convincing evidence. We have previously reported a human breast epithelial cell type (Type 1 HBEC) with stem cell characteristics and ERα (oestrogen receptor α) expression. A tumorigenic cell line, M13SV1R2, was developed from this cell type after SV40 (simian virus 40) large T‐antigen transfection and X‐ray irradiation. The cell line, however, was not responsive to oestrogen for cell growth or tumour development. In the present study, we tested the hypothesis that deprivation of growth factors and hormones may change the tumorigenicity and oestrogen response of this cell line. Results. The M13SV1R2 cells lost their tumorigenicity after culturing in a growth factor/hormone‐deprived medium for >10 passages (referred to as R2d cells) concomitant with the expression of two tumour suppressor genes, namely those coding for maspin and α6 integrin. However, these cells acquired oestrogen responsiveness in cell growth and tumour development. By immunocytochemistry, Western blotting and flow cytometry analysis, oestrogen treatment of R2d cells was found to induce many important effects related to breast carcinogenesis, namely: (i) the emergence of a subpopulation of cells expressing CD44^+/high/CD24^?/low breast tumour stem cell markers; (ii) the induction of EMT (epithelial‐to‐mesenchymal transition); (iii) the acquisition of metastatic ability; and (iv) the expression of COX‐2 (cyclo‐oxygenase‐2) through a CD44‐mediated mechanism. Conclusion. An oestrogen‐responsive cell line with ERα and CD44⁺/CD24^?/low expression can be derived from breast epithelial stem cells. The tumorigenicity and oestrogen response of these cells could depend on the cell culture conditions. The findings of this study have implications in regard to the origins of (1) ERα‐positive breast cancers, (2) CD44⁺/CD24^?/low breast tumour stem cells and (3) the metastatic ability of breast cancer.     

Retinol conversion to retinoic acid is impaired in breast cancer cell lines relative to normal cells

The bioactivity of retinol (vitamin A) is in part dependent on its metabolism to retinoic acid (RA). We investigated the ability of breast epithelial cells to synthesize RA when challenged with a physiological retinol dose (2 microM). Normal human mammary epithelial cells (HMEC) cultured from reduction mammoplasties were competent in RA synthesis and the ability to synthesize RA was retained by immortal, nontumorigenic breast epithelial cell lines (MTSV1.7, MCF-10F, and 184B5). In contrast, most (five of six) breast cancer cell lines could not synthesize RA or did so at low rates relative to normal cells. A notable exception was the MDA-MB-468 cell line, which was fully competent in RA synthesis. Most (>/=68%) of the RA synthesized by breast cells was recovered from the culture medium. Cellular retinol binding protein and cellular RA binding protein II, both expressed in HMEC, had various expression patterns in the cell lines that did not correlate with the observed differences in RA synthesizing ability. Strong RA induction of the RA hydroxylase P450RAI (CYP26) was confined to ERalpha-positive T47D and MCF-7 breast cancer cells and did not appear to explain the lack of detectable RA levels in these cells since RA remained undetectable when the cells were treated with 5-10 microM liarozole, a P450RAI inhibitor. We hypothesize that retinol bioactivity is impaired in breast cancer cells that cannot synthesize RA. In preliminary support of this hypothesis, we found that retinol (0.5-2 microM) inhibited MCF-10F but not T47D or MCF-7 cell growth.     

Involvement of p53 in gemcitabine mediated cytotoxicity and radiosensitivity in breast cancer cell lines

Gemcitabine (2',2'-difluoro-2'-deoxycytidine; dFdCyd) is one of the anti-metabolites drugs that target DNA replication. We evaluated dFdCyd cytotoxicity and its radiosensitizing ability in human breast cancer cell lines, MCF-7 (wild-type p53) and MDA-MB-231 (mutant-type p53) along with normal mammary epithelial cell line (MCF-12) for comparison. Radiosensitivity and cytotoxicity were measured by the clonogenic survival assays. DNA DSBs was studied by Pulse Field Gel Electrophoresis (PFGE) and cell cycle distribution was analyzed by flow cytometry. MDA-MB-231 cells were the most sensitive to the cytotoxicity of dFdCyd (IC(50) 5 nM) then MCF-7 (IC(50) 10nM), whereas MCF-12 cells were the most resistant to the cytotoxicity of dFdCyd (IC(50) 70 nM). MCF-12 and MCF-7 cell lines did not show any radiosensitization to dFdCyd, whereas the MDA-MB-231 cells showed significantly increased radioresistant to dFdCyd at equimolar concentration (p=0.002) and at IC(50) concentration (p<0.001). The DNA double strand breaks (DSBs) repair showed that dFdCyd neither increases DNA DSBs nor decreases the rate of their repair in MCF-12 and MCF-7 cell lines, while the same treatment in MDA-MB-231 cell line led to decrease the rate of DSBs or increase the rate of DNA repair (p=0.034). Therefore, dFdCyd is a cytotoxic agent, especially in the cancer cells irrespective of having wild-type or mutated p53 protein, but it is not effective as radiosensitizer in the cell lines used in this study. dFdCyd combined with radiation reduces the efficacy of chemo-radiotherapy in p53 mutated cells. Therefore, p53-mutated cancer could be a counter-indication for radiation-gemcitabine combined treatment.     

Expression of plasma membrane calcium pump isoform mRNAs in breast cancer cell lines

The plasma membrane Ca²⁺ ATPase (PMCA) is an important regulator of free intracellular calcium, with dynamic regulation in the rat mammary gland during lactation. Recent studies suggest that Ca²⁺ plays a role in cellular proliferation. To determine if PMCA expression is altered in tumorigenesis, we compared relative levels of PMCA1 mRNA. We found that the relative expression of PMCA1 mRNA is increased, by approximately 270% and 170%, in MCF-7 and MDA-MB-231 human breast cancer cell lines deprived of serum for 72 h, respectively, compared to the similarly treated MCF-10A human mammary gland epithelial cell line. Characterization of PMCA mRNA isoforms revealed that PMCA1b and PMCA4 mRNA are expressed in MCF-7, MDA-MB-231, SK-BR-3, ZR-75-1 and BT-483 breast cancer cell lines. We also detected PMCA2 mRNA expression in all the breast cancer cell lines examined. However, PMCA3 mRNA was only detected in BT-483 cells. Our results suggest that PMCA expression may be altered in breast cancer cell lines, suggesting altered Ca²⁺ regulation in these cell lines. Our results also indicate that breast cancer cell lines can express mRNAs for a variety PMCA isoforms.     

LncRNA ITGA9-AS1抑制乳腺癌细胞增殖并增强其对顺铂的敏感性

长链非编码RNAs (long non-coding RNAs, lncRNAs) 是一类长度大于200 nt,无蛋白质编码功能的RNAs。近年来,lncRNAs在肿瘤发生发展中的作用备受关注。LncRNAs芯片分析结合后期实时荧光定量PCR验证发现,ITGA9-AS1在MCF-7细胞中的表达量显著高于耐药细胞MCF-7/5Fu,且其在乳腺癌细胞中的表达量显著低于正常乳腺上皮细胞。生物信息学预测,ITGA9 AS1无蛋白质编码功能。在乳腺癌细胞T47D中过表达ITGA9-AS1,可显著抑制该细胞的增殖和克隆形成能力,增加该细胞对化疗药物顺铂（cisplatin, cDDP）的敏感性。相反,在乳腺上皮细胞MCF-10A中敲低ITGA9-AS1的表达,能够明显增加该细胞的增殖能力和克隆形成能力,同时降低该细胞对cDDP的敏感性。总之,lncRNA ITGA9-AS1可抑制乳腺癌细胞增殖,增强乳腺癌细胞对化疗药物的敏感性。     

2-DE analysis of a new human cell line EM-G3 derived from breast cancer progenitor cells and comparison with normal mammary epithelial cells

We performed a 2-DE analysis of proteins of the newly established spontaneously immortalized clonal cell line EM-G3 derived from a primary lesion of infiltrating ductal breast carcinoma. EM-G3 cells may represent progenitors of the mammary epithelial cells spontaneously immortalized in early phase of cancerogenesis. We compared the protein profile of EM-G3 line with proteins from populations of normal mammary epithelial cells (NME), and determined the phenotype of both types of cells. NME cells are a mixture of both main cell types in breast epithelia, myoepithelial and luminal cells. The EM-G3 breast cancer cell line has a unique basal-like phenotype. We identified proteins that are differently expressed in these cells. Cytokeratin 16, cytokeratin 19, squamous cell carcinoma antigen 1, caphepsin B and caspase 14 were predominantly expressed by NME cells. Cytokeratin 13, isoelectric variant of annexin 5, isoelectric variant of chloride intracellular channel protein 1, glyoxalase 1 and glutamine synthetase were predominantly expressed by EM-G3 cells. The proteins up-regulated in EM-G3 cells may represent potential protein markers of mammary epithelial cells progenitors and may be important in early phase of carcinogenesis.     

呼吸道苦味信号转导抑制与小鼠哮喘相关

鉴于哮喘病患病人数众多,约有一半的病人病情得不到较好的控制,急需新的治疗方法和药物.最近研究发现,苦味受体(bitter taste receptors,T2 Rs)在多个组织中表达,且苦味剂对哮喘有治疗潜力,T2Rs有可能成为哮喘治疗的新靶点.本文选C57BL/6小鼠随机分为对照组、二氧化硫(sulfur dioxi...     

Insulin-like growth factor binding protein-related protein 1 inhibits proliferation of MCF-7 breast cancer cells via a senescence-like mechanism.

Elevated insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) mRNA in senescent human mammary epithelial cells suggested that the IGFBP-3 gene product may inhibit cell proliferation. To test this hypothesis, we used a retroviral vector to express IGFBP-rP1 cDNA in the IGFBP-rP1-deficient MCF-7 breast cancer cell line. Compared with control vector-transduced cells, cumulative cell numbers for IGFBP-rP1-transduced polyclonal or clonal cell cultures were reduced by 39 and 74%, respectively, after 1 week. Medium conditioned by IGFBP-rP1-producing cultures reduced cumulative cell numbers in parental MCF-7 cultures by 20% compared with medium from cultures of a control vector-transduced cell line. Nuclear fragmentation analysis and cell proliferation assays completed in the presence of the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone excluded apoptosis as the responsible mechanism. The percentage of cells containing senescence-associated beta-galactosidase activity was doubled compared with control cell cultures. Flow cytometry analysis indicated that twice as many noncycling cells were present in the IGFBP-rP1-transduced MCF-7 cell cultures compared with controls. These findings indicate that IGFBP-rP1 is an inhibitor of MCF-7 breast cancer cell proliferation and may act via a cellular senescence-like mechanism.     

Epigenetic regulation of bone morphogenetic protein-6 gene expression in breast cancer cells

Bone morphogenetic protein-6 (BMP-6) is closely correlated with tumor differentiation and skeletal metastasis. Our previous research found that BMP-6 gene expression can be activated dose-dependently by estrogen in estrogen receptor positive (ER⁺) breast cancer cell line MCF-7, but not in ER negative (ER⁻) cell line MDA-MB-231. This experiment is designed to investigate the epigenetic regulatory mechanism of the BMP-6 gene expression in breast cancer cell lines MDA-MB-231, MCF-7 and T47D with regard to the methylation status in the 5′ flanking region of the human BMP-6 gene. The endogenous level of BMP-6 mRNA in ER⁻ cell line MDA-MB-231 was relatively lower than that in ER⁺ MCF-7 and T47D cell lines. After the treatment with 5-aza-2′-deoxycytidine (5-aza-dC, especially in the concentration of 10 μM), the BMP-6 mRNA expression in MDA-MB-231 was obviously up-regulated. However, 5-aza-dC treatment failed to regulate the expression of BMP-6 in MCF-7 and T47D cells. Using enzyme restriction PCR (MSRE-PCR), as well as bisulfite sequencing (BSG), methylation of human BMP-6 gene promoter was detected in MDA-MB-231; while in MCF-7 and T47D, BMP-6 gene promoter remained demethylated status. In 33 breast tumor specimens, promoter methylation of BMP-6 was detected by methylation-specific PCR, hypermethylation of BMP-6 was observed in ER negative cases (16 of 16 cases (100%)), while obviously lower methylation frequency were observed in ER positive cases (3 of 17 cases (18%)), indicating that BMP-6 promoter methylation status is correlated with ER status in breast cancer.     

The 5-HT2A serotoninergic receptor is expressed in the MCF-7 human breast cancer cell line and reveals a mitogenic effect of serotonin

Serotonin (5-hydroxytryptamine, 5-HT) has been described as a mitogen in a variety of cell types and carcinomas. It exerts its mitogenic effect by interacting with a wide range of 5-HT receptor types. Certain studies suggest that some selective serotonin re-uptake inhibitors promote breast cancer in animals and humans. This study attempts to clarify the role of serotonin in promoting the growth of neoplastic mammary cells. Expression of the 5-HT(2A) serotoninergic receptor subtype in MCF-7 cells was determined by RT-PCR, Western blotting, and immunofluorescence analysis. The mitogenic effect of 5-HT on MCF-7 cells was determined by means of the MTT proliferation assay. We have demonstrated that the 5-HT(2A) receptor subtype is fully expressed in the MCF-7 human breast cancer cell line, in terms of encoding mRNA and receptor protein. Automated sequencing has confirmed that the 5-HT(2A) receptor present in this cell line is identical to the 5-HT(2A) receptor found in human platelets and in human cerebral cortex. Furthermore, this receptor was found by immunofluorescence to be on the plasma membrane. MTT proliferation assays revealed that 5-HT and DOI, a selective 5-HT(2A) receptor subtype agonist, stimulated MCF-7 cell. These results indicate that 5-HT plays a mitogenic role in neoplastic mammary cells. Our data also indicate that 5-HT exerts this positive growth effect on MCF-7 cells through, in part, the 5-HT(2A) receptor subtype, which is fully expressed in this cell line.     

Leptin mediates a proliferative response in human MCF7 breast cancer cells

Obesity is a risk factor of breast cancers. As leptin, a hormone mainly secreted by white adipocytes, elicits proliferative effects in some cell types, we tested the hypothesis that leptin could influence human breast cancer MCF-7 cell growth. Here we show that MCF-7 cells express leptin receptors and respond to human recombinant leptin by STAT3 and p42/p44 MAPkinase activations and by increased proliferation. These findings suggest that leptin could act in vivo as a paracrine/endocrine growth factor towards mammary epithelial cells thus contributing to explain why obesity is a risk factor of developing breast cancers.     

Two families of candidate taste receptors in fishes

Vertebrates receive tastants, such as sugars, amino acids, and nucleotides, via taste bud cells in epithelial tissues. In mammals, two families of G protein-coupled receptors for tastants are expressed in taste bud cells-T1Rs for sweet tastants and umami tastants (l-amino acids) and T2Rs for bitter tastants. Here, we report two families of candidate taste receptors in fish species, fish T1Rs and T2Rs, which show significant identity to mammalian T1Rs and T2Rs, respectively. Fish T1Rs consist of three types: fish T1R1 and T1R3 that show the highest degrees of identity to mammalian T1R1 and T1R3, respectively, and fish T1R2 that shows almost equivalent identity to both mammalian T1R1 and T1R2. Unlike mammalian T1R2, fish T1R2 consists of two or three members in each species. We also identified two fish T2Rs that show low degrees of identity to mammalian T2Rs. In situ hybridization experiments revealed that fish T1R and T2R genes were expressed specifically in taste bud cells, but not in olfactory receptor cells. Fish T1R1 and T1R2 genes were expressed in different subsets of taste bud cells, and fish T1R3 gene was co-expressed with either fish T1R1 or T1R2 gene as in the case of mammals. There were also a significant number of cells expressing fish T1R2 genes only. Fish T2R genes were expressed in different cells from those expressing fish T1R genes. These results suggest that vertebrates commonly have two kinds of taste signaling pathways that are defined by the types of taste receptors expressed in taste receptor cells.     

Adiponectin mediates antiproliferative and apoptotic responses in human MCF7 breast cancer cells

It is well established that obesity is a risk factor for breast cancer and that blood levels of adiponectin, a hormone mainly secreted by white adipocytes, are inversely correlated with the body fat mass. As adiponectin elicits anti-proliferative effects in some cell types, we tested the hypothesis that adiponectin could influence human breast cancer MCF-7 cell growth. Here we show that MCF-7 cells express adiponectin receptors and respond to human recombinant adiponectin by reducing their growth, AMPkinase activation, and p42/p44 MAPkinase inactivation. Further, we demonstrate that the anti-proliferative effect of adiponectin involves activation of cell apoptosis and inhibition of cell cycle. These findings suggest that adiponectin could act in vivo as a paracrine/endocrine growth inhibitor towards mammary epithelial cells. Moreover, adipose adiponectin production being strongly reduced in obesity, this study may help to explain why obesity is a risk factor of developing breast cancers.     

In vitro selection of aptamer S1 against MCF-7 human breast cancer cells

Breast cancer is the most common female cancer. However, the known effective specific biomarkers for breast cancer are still scarce. Abnormal membrane proteins serve as ideal biomarkers for disease diagnoses, therapeutics and prognosis. Thus aptamers (single-stranded oligonucleotide molecules) with molecular recognition properties can be used as efficient tools to sort cells based on differences in cell surface architecture between normal and tumor cells. In this study, we aimed to screen specific aptamer against MCF-7 human breast cancer cells. Cell-SELEX process was performed to isolate aptamers from a combinatorial single-stranded nucleic acid library that selectively targeting surface proteins of MCF-7 cells in contrast with MCF-10A human mammary epithelial cells. The process was repeated until the pool was enriched for sequences that specifically recognizing MCF-7 cells in monitoring by flow cytometry. Subsequently, the enriched pool was cloned into bacteria, and positive clones were sequenced to obtain individual sequences. Representative sequences were chemically synthesized and evaluated their binding affinities to MCF-7 cells. As a result, an aptamer S1 was finally identified to have high binding affinity with equilibrium dissociation constant (K_d) value of 29.9 ± 6.0 nM. FAM-labeled aptamer S1 induced fluorescence shift in MCF-7 cells but not in MCF-10A human mammary epithelial cells, or MDA-MB-453 and MDA-MB-231 human breast cancer cells. Furthermore, result of cell imaging observed from laser confocal fluorescence microscope showed that MCF-7 cells exhibited stronger fluorescence signal resulted from Cy5-labeled aptamer S1 than MCF-10A cells. The above findings suggested that S1 may be a specificity and selectivity aptamer for MCF-7 cells and useful for the breast cancer detection and diagnosis.     

Specific expression of the human voltage-gated proton channel Hv1 in highly metastatic breast cancer cells, promotes tumor progression and metastasis

The newly discovered human voltage-gated proton channel Hv1 is essential for proton transfer, which contains a voltage sensor domain (VSD) without a pore domain. We report here for the first time that Hv1 is specifically expressed in the highly metastatic human breast tumor tissues, but not in poorly metastatic breast cancer tissues, detected by immunohistochemistry. Meanwhile, real-time RT-PCR and immunocytochemistry showed that the expression levels of Hv1 have significant differences among breast cancer cell lines, MCF-7, MDA-MB-231, MDA-MB-468, MDA-MB-453, T-47D and SK-BR-3, in which Hv1 is expressed at a high level in highly metastatic human breast cancer cell line MDA-MB-231, but at a very low level in poorly metastatic human breast cancer cell line MCF-7. Inhibition of Hv1 expression in the highly metastatic MDA-MB-231 cells by small interfering RNA (siRNA) significantly decreases the invasion and migration of the cells. The intracellular pH of MDA-MB-231 cells down-regulated Hv1 expression by siRNA is obviously decreased compared with MDA-MB-231 with the scrambled siRNA. The expression of matrix metalloproteinase-2 and gelatinase activity in MDA-MB-231 cells suppressed Hv1 by siRNA were reduced. Our results strongly suggest that Hv1 regulates breast cancer intracellular pH and exacerbates the migratory ability of metastatic cells.