MiR-29b may suppresses peritoneal metastases through inhibition of the mesothelial–mesenchymal transition (MMT) of human peritoneal mesothelial cells

Peritoneal dissemination is a major metastatic pathway for gastrointestinal and ovarian malignancies. The miR-29b family is downregulated in peritoneal fluids in patients with peritoneal metastases (PM). We examined the effect of miR-29b on mesothelial cells (MC) which play critical a role in the development of PM through mesothelial-mesenchymal transition (MMT). Human peritoneal mesothelial cells (HPMCs) were isolated from surgically resected omental tissue and MMT induced by stimulation with 10 ng/ml TGF-β1. MiR-29b mimics and negative control miR were transfected by lipofection using RNAiMAX and the effects on the MMT evaluated in vitro. HPMC produced substantial amounts of miR-29b which was markedly inhibited by TGF-β1. TGF-β1 stimulation of HPMC induced morphological changes with decreased expression of E-cadherin and calretinin, and increased expression of vimentin and fibronectin. TGF-β1 also enhanced proliferation and migration of HPMC as well as adhesion of tumor cells in a fibronectin dependent manner. However, all events were strongly abrogated by simultaneous transfection of miR-29b. MiR-29b inhibits TGF-β1 induced MMT and replacement of miR-29b in the peritoneal cavity might be effective to prevent development of PM partly through the effects on MC.

Astragalus Total Saponins Ameliorate Peritoneal Fibrosis by Promoting Mitochondrial Synthesis and Inhibiting Apoptosis

Peritoneal fibrosis (PF) is a disease caused by prolonged exposure of the peritoneum to high levels of dialysis fluid. Astragalus total saponins (ATS) is a phytochemical naturally occurring in Radix Astragali that has anti-inflammatory and anti-oxidant properties. In this study, we constructed an in vivo model of PF using 4.25% glucose-containing administered intraperitoneally to rats and incubated peritoneal mesothelial cells (PMCs) with 4.25% glucose-containing peritoneal dialysis fluid to construct an in vitro model of PF. Furthermore, siRNA of PGC-1[Formula: see text] was used to inhibit the expression of PGC-1[Formula: see text] to further investigate the mechanism of the protective effect of ATS on PF. In both in vivo and in vitro models, ATS treatment showed a protective effect against PF, with ATS reducing the thickness of peritoneal tissues in PF rats, increasing the viability of PMCs, increasing the mitochondrial membrane potential and reducing apoptosis ratio. ATS treatment also reduced the expressions of peritoneal fibrosis markers (Smad2, p-Smad2 and [Formula: see text]-SMA) and apoptosis markers (Caspase3, cleaved-Caspase3 and Bax) and restored the expressions of mitochondrial synthesis proteins (PGC-1[Formula: see text], NRF1 and TFAM) in ATS-treated peritoneal tissues or PMCs. Furthermore, in the presence of PGC-1[Formula: see text] inhibition, the protective effect of ATS on PF was blocked. In conclusion, ATS treatment may be an effective therapeutic agent to inhibit high glucose-induced in peritoneal fibrosis through PGC-1[Formula: see text]-mediated apoptosis.

Amelioration of human peritoneal mesothelial cell co-culture-evoked malignant potential of ovarian cancer cells by acacetin involves LPA release-activated RAGE-PI3K/AKT signaling

Background Ovarian cancer is a devastating gynecological malignancy and frequently presents as an advanced carcinoma with disseminated peritoneum metastasis. Acacetin exerts anti-cancerous effects in several carcinomas. Here, we sought to investigate acacetin function in ovarian cancer malignancy triggered by peritoneal mesothelial cells. Methods Peritoneal mesothelial cells were treated with acacetin, and then the conditioned medium was collected to treat ovarian cancer cells. Then, cell proliferation was analyzed by MTT assay. Transwell analysis was conducted to evaluate cell invasion. Protein expression was determined by western blotting. ELISA and qRT-PCR were applied to analyze inflammatory cytokine levels. The underlying mechanism was also explored. Results Acacetin suppressed cell proliferation and invasion, but enhanced cell apoptosis. Furthermore, mesothelial cell-evoked malignant characteristics were inhibited when mesothelial cells were pre-treated with acacetin via restraining cell proliferation and invasion, concomitant with decreases in proliferation-related PCNA, MMP-2 and MMP-9 levels. Simultaneously, acacetin reduced mesothelial cell-induced transcripts and production of pro-inflammatory cytokine IL-6 and IL-8 in ovarian cancer cells. Mechanically, acacetin decreased lysophosphatidic acid (LPA) release from mesothelial cells, and subsequent activation of receptor for advanced glycation end-products (RAGE)-PI3K/AKT signaling in ovarian cancer cells. Notably, exogenous LPA restored the above pathway, and offset the efficacy of acacetin against mesothelial cell-evoked malignancy in ovarian cancer cells, including cell proliferation, invasion and inflammatory cytokine production. Conclusions Acacetin may not only engender direct inhibition of ovarian cancer cell malignancy, but also antagonize mesothelial cell-evoked malignancy by blocking LPA release-activated RAGE-PI3K/AKT signaling. Thus, these findings provide supporting evidence for a promising therapeutic agent against ovarian cancer. Graphical Abstract

Three newly established immortalized mesothelial cell lines exhibit morphological phenotypes corresponding to malignant mesothelioma epithelioid, intermediate, and sarcomatoid types, respectively

Background Malignant mesothelioma (MM) is a very aggressive tumor that develops from mesothelial cells, mainly due to asbestos exposure. MM is categorized into three major histological subtypes: epithelioid, sarcomatoid, and biphasic, with the biphasic subtype containing both epithelioid and sarcomatoid components. Patients with sarcomatoid mesothelioma usually show a poorer prognosis than those with epithelioid mesothelioma, but it is not clear how these morphological phenotypes are determined or changed during the oncogenic transformation of mesothelial cells. Methods We introduced the E6 and E7 genes of human papillomavirus type 16 and human telomerase reverse transcriptase gene in human peritoneal mesothelial cells and established three morphologically different types of immortalized mesothelial cell lines. Results HOMC-B1 cells exhibited epithelioid morphology, HOMC-A4 cells were fibroblast-like, spindle-shaped, and HOMC-D4 cells had an intermediate morphology, indicating that these three cell lines closely mimicked the histological subtypes of MM. Gene expression profiling revealed increased expression of NOD-like receptor signaling-related genes in HOMC-A4 cells. Notably, the combination treatment of HOMC-D4 cells with TGF-β and IL-1β induced a morphological change from intermediate to sarcomatoid morphology. Conclusions Our established cell lines are useful for elucidating the fundamental mechanisms of mesothelial cell transformation and mesothelial-to-mesenchymal transition.

Comparison of Isotonic Activation of Cell Volume Regulation in Rat Peritoneal Mesothelial Cells and in Kidney Outer Medullary Collecting Duct Principal Cells

In disease states, mesothelial cells are exposed to variable osmotic conditions, with high osmotic stress exerted by peritoneal dialysis (PD) fluids. They contain unphysiologically high concentrations of glucose and result in major peritoneal membrane transformation and PD function loss. The effects of isotonic entry of urea and myo-inositol in hypertonic (380 mOsm/kg) medium on the cell volume of primary cultures of rat peritoneal mesothelial cells and rat kidney outer medullary collecting duct (OMCD) principal cells were studied. In hypertonic medium, rat peritoneal mesothelial cells activated a different mechanism of cell volume regulation in the presence of isotonic urea (100 mM) in comparison to rat kidney OMCD principal cells. In kidney OMCD cells inflow of urea into the shrunken cell results in restoration of cell volume. In the shrunken peritoneal mesothelial cells, isotonic urea inflow caused a small volume increase and activated regulatory volume decrease (RVD). Isotonic myo-inositol activated RVD in hypertonic medium in both cell types. Isotonic application of both osmolytes caused a sharp increase of intracellular calcium both in peritoneal mesothelial cells and in kidney OMCD principal cells. In conclusion, peritoneal mesothelial cells exhibit RVD mechanisms when challenged with myo-inositol and urea under hyperosmolar isotonic switch from mannitol through involvement of calcium-dependent control. Myo-inositol effects were identical with the ones in OMCD principal cells whereas urea effects in OMCD principal cells led to no RVD induction.

Cellular Integrin α5β1 and Exosomal ADAM17 Mediate the Binding and Uptake of Exosomes Produced by Colorectal Carcinoma Cells

Approximately 25% of colorectal cancer (CRC) patients develop peritoneal metastasis, a condition associated with a bleak prognosis. The CRC peritoneal dissemination cascade involves the shedding of cancer cells from the primary tumor, their transport through the peritoneal cavity, their adhesion to the peritoneal mesothelial cells (PMCs) that line all peritoneal organs, and invasion of cancer cells through this mesothelial cell barrier and underlying stroma to establish new metastatic foci. Exosomes produced by cancer cells have been shown to influence many processes related to cancer progression and metastasis. In epithelial ovarian cancer these extracellular vesicles (EVs) have been shown to favor different steps of the peritoneal dissemination cascade by changing the functional phenotype of cancer cells and PMCs. Little is currently known, however, about the roles played by exosomes in the pathogenesis and peritoneal metastasis cascade of CRC and especially about the molecules that mediate their interaction and uptake by target PMCs and tumor cells. We isolated exosomes by size−exclusion chromatography from CRC cells and performed cell-adhesion assays to immobilized exosomes in the presence of blocking antibodies against surface proteins and measured the uptake of fluorescently-labelled exosomes. We report here that the interaction between integrin α5β1 on CRC cells (and PMCs) and its ligand ADAM17 on exosomes mediated the binding and uptake of CRC-derived exosomes. Furthermore, this process was negatively regulated by the expression of tetraspanin CD9 on exosomes.

Thalidomide Suppresses Angiogenesis Through the Signal Transducer and Activator of Transcription 3/SP4 Signaling Pathway in the Peritoneal Membrane

Peritoneal angiogenesis is the key pathophysiological factor that limits peritoneal ultrafiltration during peritoneal dialysis (PD) in uremic patients. Thalidomide has been confirmed to inhibit angiogenesis by inhibiting the secretion of vascular endothelial growth factor (VEGF), but the exact mechanism by which thalidomide inhibits vascular proliferation during PD is still unclear. Here, the objective of the present study was to investigate whether the reduction in VEGF production by human peritoneal mesothelial cells (HPMCs) was controlled by thalidomide. Stimulation of HPMCs with IL-6 in combination with soluble IL-6 receptor (sIL-6R) promoted VEGF expression and secretion, but these effects were attenuated by thalidomide treatment through a transcriptional mechanism that involved signal transducer and activator of transcription 3 (STAT3) and SP4. Conditioned medium from HPMCs cultured with thalidomide inhibited angiogenic endothelial tube formation, which could be further blocked by silencing SP4 and promoted by overexpressing SP4. In vivo, induction of peritoneal angiogenesis in sham rats, sham+PD rats, 5/6 nephrectomy (5/6Nx) rats, 5/6Nx+PD rats, and 5/6Nx+PD rats intraperitoneally treated with thalidomide showed that thalidomide was involved in the control of several key endothelial–specific targets, including VEGFR2, VEGFR3, SP4, and STAT3 expression and new vessel formation, confirming the role of thalidomide and STAT3/SP4 signaling in these processes. Taken together, these findings identify a novel mechanism that links thalidomide, STAT3/SP4 signaling, and angiogenesis in the peritoneal membrane.