The anti-tumoral effects of the oxygen carrier YQ23 in a triple-negative breast cancer syngeneic model.

BACKGROUND: Triple-negative breast cancer (TNBC), an aggressive breast cancer subtype, is associated with poor prognosis and high mortality rate. In the search for effective therapeutic options, preclinical studies have suggested using systemic oxygenation to inhibit tumor growth and metastasis in various cancer models, including TNBC, by weakening the hypoxia-A2A adenosine receptors (A2AR)-driven immunosuppression in the tumor microenvironment (TME). In our present study, a hemoglobin-based oxygen carrier (HBOC) "YQ23" was tested for its role in modulating the TME and tumor inhibition. METHODS: A syngeneic TNBC mouse model was established by inoculating 4T1 cells subcutaneously in BALB/c mice. Tumor (~100 mm3) bearing mice were treated either with saline or YQ23 (400 mg/kg) i.v. once weekly. To prove the immune-regulatory role of YQ23, CD4+ and CD8+ cells were depleted from a group of mice prior to treatment. Tumor growth was monitored for four weeks while xenografts were isolated at the end of the treatment for ex vivo immunohistological examination. RESULTS: YQ23 significantly inhibited the tumor growth, and this suppressive effect was abolished by depleting the host immune cells. Immunohistochemical staining of xenograft sections showed YQ23 reduced the level of hypoxia and adenosine producing ecto-enzyme CD73. Although there was no significant difference in the make up of the intra-tumoral immune populations, we observed a down-regulation of the immune checkpoint PD-1. In concordance with the weakened immunosuppression, the inflammatory cytokine interferon γ and cytolytic granzyme B were upregulated. CONCLUSIONS: YQ23 treatment may be a potential therapeutic strategy to modulate the TME in TNBC.

Oxygen carrier YQ23 can enhance the chemotherapeutic drug responses of chemoresistant esophageal tumor xenografts.

PURPOSE: Adjunct chemoradiation is offered to unresectable esophageal squamous cell carcinoma (ESCC) patients, while its use is limited in tumors with strong resistance. Oxygen carriers or anti-hypoxic drugs belong to an emerging class of regulators that can alleviate tumor hypoxia. METHODS: We investigate the potential use of a novel oxygen carrier YQ23 in sensitizing chemoresistant ESCC in a series of subcutaneous tumor xenograft models developed using ESCC cell lines with different strengths of chemosensitivities. RESULTS: Tumor xenografts were developed using SLMT-1 and HKESC-2 ESCC cell lines with different strengths of resistance to two chemotherapeutic drugs, 5-fluorouracil and cisplatin. More resistant SLMT-1 xenografts responded better to YQ23 treatment than HKESC-2, as reflected by the induced tumor oxygen level. YQ23 sensitized SLMT-1 xenografts toward 5-fluorouracil via its effect on reducing the level of a hypoxic marker HIF-1α. Furthermore, a derangement of tumor microvessel density and integrity was demonstrated with a concurrent decrease in the level of a tumor mesenchymal marker vimentin. Similar to the 5-fluorouracil sensitizing effect, YQ23 also enhanced the response of SLMT-1 xenografts toward cisplatin by reducing the tumor size and the number of animals with invasive tumors. Chemosensitive HKESC-2 xenografts were irresponsive to combined YQ23 and cisplatin treatment. CONCLUSIONS: In all, YQ23 functions selectively on chemoresistant ESCC xenografts, which implicates its potential use as a chemosensitizing agent for ESCC patients.

The estrogen-related receptor alpha upregulates secretin expressions in response to hypertonicity and angiotensin II stimulation.

Osmoregulation via maintenance of water and salt homeostasis is a vital process. In the brain, a functional secretin (SCT) and secretin receptor (SCTR) axis has recently been shown to mediate central actions of angiotensin II (ANGII), including initiation of water intake and stimulation of vasopressin (VP) expression and release. In this report, we provide evidence that estrogen-related receptor α (ERRα, NR3B1), a transcription factor mainly involved in metabolism, acts as an upstream activator of the SCT gene. In vitro studies using mouse hypothalamic cell line N-42 show that ERRα upregulates SCT promoter and gene expression. More importantly, knockdown of endogenous ERRα abolishes SCT promoter activation in response to hypertonic and ANGII stimulations. In mouse brain, ERRα coexpresses with SCT in various osmoregulatory brain regions, including the lamina terminalis and the paraventricular nucleus of the hypothalamus, and its expression is induced by hyperosmotic and ANGII treatments. Based on our data, we propose that both the upregulation of ERRα and/or the increased binding of ERRα to the mouse SCT promoter are two possible mechanisms for the elevated SCT expression upon hyperosmolality and central ANGII stimulation.

An indispensable role of secretin in mediating the osmoregulatory functions of angiotensin II.

Fluid balance is critical to life and hence is tightly controlled in the body. Angiotensin II (ANGII), one of the most important components of this regulatory system, is recognized as a dipsogenic hormone that stimulates vasopressin (VP) expression and release. However, detailed mechanisms regarding how ANGII brings about these changes are not fully understood. In the present study, we show initially that the osmoregulatory functions of secretin (SCT) in the brain are similar to those of ANGII in mice and, more important, we discovered the role of SCT as the link between ANGII and its downstream effects. This was substantiated by the use of two knockout mice, SCTR(-/-) and SCT(-/-), in which we show the absence of an intact SCT/secretin receptor (SCTR) axis resulted in an abolishment or much reduced ANGII osmoregulatory functions. By immunohistochemical staining and in situ hybridization, the proteins and transcripts of SCT and its receptor are found in the paraventricular nucleus (PVN) and lamina terminalis. We propose that SCT produced in the circumventricular organs is transported and released in the PVN to stimulate vasopressin expression and release. In summary, our findings identify SCT and SCTR as novel elements of the ANGII osmoregulatory pathway in maintaining fluid balance in the body.

Knockout of secretin receptor reduces large cholangiocyte hyperplasia in mice with extrahepatic cholestasis induced by bile duct ligation.

UNLABELLED: During bile duct ligation (BDL), the growth of large cholangiocytes is regulated by the cyclic adenosine monophosphate (cAMP)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and is closely associated with increased secretin receptor (SR) expression. Although it has been suggested that SR modulates cholangiocyte growth, direct evidence for secretin-dependent proliferation is lacking. SR wild-type (WT) (SR(+/+)) or SR knockout (SR(-/-)) mice underwent sham surgery or BDL for 3 or 7 days. We evaluated SR expression, cholangiocyte proliferation, and apoptosis in liver sections and proliferating cell nuclear antigen (PCNA) protein expression and ERK1/2 phosphorylation in purified large cholangiocytes from WT and SR(-/-) BDL mice. Normal WT mice were treated with secretin (2.5 nmoles/kg/day by way of osmotic minipumps for 1 week), and biliary mass was evaluated. Small and large cholangiocytes were used to evaluate the in vitro effect of secretin (100 nM) on proliferation, protein kinase A (PKA) activity, and ERK1/2 phosphorylation. SR expression was also stably knocked down by short hairpin RNA, and basal and secretin-stimulated cAMP levels (a functional index of biliary growth) and proliferation were determined. SR was expressed by large cholangiocytes. Knockout of SR significantly decreased large cholangiocyte growth induced by BDL, which was associated with enhanced apoptosis. PCNA expression and ERK1/2 phosphorylation were decreased in large cholangiocytes from SR(-/-) BDL compared with WT BDL mice. In vivo administration of secretin to normal WT mice increased ductal mass. In vitro, secretin increased proliferation, PKA activity, and ERK1/2 phosphorylation of large cholangiocytes that was blocked by PKA and mitogen-activated protein kinase kinase inhibitors. Stable knockdown of SR expression reduced basal cholangiocyte proliferation. SR is an important trophic regulator sustaining biliary growth. CONCLUSION: The current study provides strong support for the potential use of secretin as a therapy for ductopenic liver diseases.

Bile acids inhibit duodenal secretin expression via orphan nuclear receptor small heterodimer partner (SHP).

Small heterodimer partner (SHP) is an orphan nuclear receptor in which gene expression can be upregulated by bile acids. It regulates its target genes by repressing the transcriptional activities of other nuclear receptors including NeuroD, which has been shown to regulate secretin gene expression. Here, we evaluated the regulation on duodenal secretin gene expression by SHP and selected bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA). In vitro treatment of CDCA or fexaramine elevated the SHP transcript level and occupancy on secretin promoter. The increase in the SHP level, induced by bile acid treatment or overexpression, reduced secretin gene expression, whereas this gene inhibitory effect was reversed by silencing of endogenous SHP. In in vivo studies, double-immunofluorescence staining demonstrated the coexpression of secretin and SHP in mouse duodenum. Feeding mice with 1% CA-enriched rodent chow resulted in upregulation of SHP and a concomitant decrease in secretin transcript and protein levels in duodenum compared with the control group fed with normal chow. A diet enriched with 5% cholestyramine led to a decrease in SHP level and a corresponding increase in secretin expression. Overall, this study showed that bile acids via SHP inhibit duodenal secretin gene expression. Because secretin is a key hormone that stimulates bile flow in cholangiocytes, this pathway thus provides a novel means to modulate secretin-stimulated choleresis in response to intraduodenal bile acids.

A functional variable number of tandem repeats is located at the 5' flanking region of the human secretin gene plays a downregulatory role in expression.

Secretin is a peptide hormone playing multiple functions in the brain-gut axis. In this report, we investigated, by promoter analysis, the potential function of the variable of tandem repeats (VNTR), located at the 5' upstream region of the human secretin gene, and we demonstrated for the first time that this VNTR could downregulate transcription of the human secretin gene in a promoter-specific manner. The efficiency of VNTR in silencing the promoter was found to be directly related the number of repetitive units residing within. We also showed the deoxyribonucleic acid sequence as well as the length polymorphism of the VNTR of 76 Chinese individuals. These results collectively suggest that VNTR could potentially be a functional regulator to control the expression of the human secretin gene in different individuals.

Multiple actions of secretin in the human body.

The discovery of secretin initiated the field of endocrinology. Over the past century, multiple gastrointestinal functions of secretin have been extensively studied, and it was discovered that the principal function of this peptide in the gastrointestinal system is to facilitate digestion and to provide protection. In view of the late identification of secretin and the secretin receptor in various tissues, including the central nervous system, the pleiotropic functions of secretin have more recently been an area of intense focus. Secretin is a classical hormone, and recent studies clearly showed secretin's involvement in neural and neuroendocrine pathways, although the neuroactivity and neural regulation of its release are yet to be elucidated. This chapter reviews our current understanding of the pleiotropic actions of secretin with a special focus on the hormonal and neural interdependent pathways that mediate these actions.

Signaling mechanisms of secretin receptor.

Secretin, a 27-amino acid gastrointestinal peptide, was initially discovered based on its activities in stimulating pancreatic juice. In the past 20 years, secretin was demonstrated to exhibit pleiotropic functions in many different tissues and more importantly, its role as a neuropeptide was substantiated. To carry out its activities in the central nervous system and in peripheral organs, secretin interacts specifically with one known receptor. Secretin receptor, a member of guanine nucleotide-binding protein (G protein)-coupled receptor (GPCR) in the secretin/VIP/glucagon subfamily, possesses the characteristics of GPCR with seven conserved transmembrane domains, a relatively large amino-terminal extracellular domain and an intracellular carboxyl terminus. The structural features and signal transduction pathways of the secretin receptor in various tissues are reviewed in this article.

Localization of small heterodimer partner (SHP) and secretin in mouse duodenal cells.

Previous studies have demonstrated the transcriptional repressive property of the atypical nuclear receptor, small heterodimer partner (SHP), on NeuroD. NeuroD is a basic helix-loop-helix transcription factor that has also been shown to be important in modulating secretin gene expression. The present study revealed the activation of the human secretin core promotor by overexpressing NeuroD, and the localization of SHP and secretin-producing cells in mouse duodenal epithelium by immunohistochemical stainings. These results indicated that SHP and secretin are potentially co-expressed and lead us to propose a novel regulatory pathway, in which SHP represses NeuroD's positive regulatory activity on secretin gene.